Research


Olfactory Stem Cells and Neural Regeneration

The generation of neuronal diversity in the nervous system requires the specification and differentiation of a multitude of cellular lineages. Successive developmental programs control the generation of individual neuronal types, cell migration, axon extension, and ultimately the formation of functional synaptic connections. The specific genetic programs underlying the differentiation of mature neurons from their progenitors remain incompletely characterized, in part because of the difficulty in studying neuronal progenitor cells in their native environments.

In the vertebrate olfactory system, primary sensory neurons are continuously regenerated throughout adult life via the proliferation and differentiation of multipotent neural progenitor cells. This feature makes the olfactory system particularly amenable for studies on adult neurogenesis and the properties of neuronal stem cells. Olfactory receptor neurons normally turn over every 30-60 days and are replaced through the proliferation and differentiation of multipotent progenitor cells: normally quiescent stem cells known as horizontal basal cells (HBCs) and proliferative progenitors known as globose basal cells (GBCs). Following injury that results in the destruction of mature cells in the olfactory epithelium, the HBCs proliferate and differentiate – using GBCs as intermediates – to reconstitute all cellular constituents of this sensory epithelium, including the olfactory receptor neurons (ORNs), sustentacular cells (Sus) and cells of the Bowman's Gland (BG). The regenerative capacity of the olfactory epithelium represents a powerful and experimentally accessible paradigm for understanding the regulation of neural stem cell function under normal conditions and during injury-induced regeneration. While distinct stages of the olfactory lineage have been identified, however, much remains to be learned about the genetic programs that both define and regulate olfactory neurogenesis during development and regeneration. Moreover, virtually nothing is known about the transcriptional networks regulating the HBCs and GBCs, the adult tissue stem cells of the postnatal olfactory epithelium.

Current projects are using a variety of approaches to elucidate the molecular and cellular mechanisms regulating olfactory stem cells and olfactory neurogenesis in the mouse. In one area of inquiry, using conditional genetic knockouts we are investigating the roles of certain transcription factors – e.g., p63 – and intracellular signaling pathways in promoting stem cell self-renewal, proliferation and differentiation. Other studies are using clonal analysis in vivo to determine how patterns of symmetric and asymmetric cell division support stem cell renewal and tissue maintenance. Finally, we are developing and applying single cell RNA-Seq technologies to elucidate the developmental trajectories of cells as they transition from early stem cell states, through intermediate progenitors and then through terminal differentiation.


Together our studies provide a model for understanding the mechanisms regulating neural stem cells and lay the groundwork for the future development of treatments and therapeutics to ameliorate tissue damage and degeneration in the nervous system.


  

Zebrafish: A Model for Studying Olfactory-Guided Innate Behavior

An understanding of the neural circuits underlying innate behaviors is required for an eventual understanding of the causes of human conditions such as chronic fear and anxiety. Many innate behaviors begin with the reception of a sensory stimulus and subsequent processing of diverse and complex inputs by the brain. In the olfactory system, pheromones can excite specific receptors in select neurons to cause a fixed action response. For example, individuals sensing danger release alarm pheromones, which elicit fear and defensive behaviors in other members of the species. Although such pheromones provide unique tools for probing sensory-guided behaviors, the higher-order neural circuitry mediating these behaviors remains obscure. We are using the zebrafish as an experimental paradigm for elucidating the networks driving sensory guided innate behaviors. Compared to mammalian species, zebrafish have a relatively simple and therefore experimentally tractable nervous system. Zebrafish exhibit an olfactory-mediated fear behavior that can be controlled by a small molecule alarm pheromone. The alarm response shows many of the hallmarks of mammalian fear behaviors, and the behavior of adult and larval zebrafish to alarm pheromone is similar to a fear response generated by other sensory inputs. Moreover, zebrafish are amenable to genetic and chemical manipulation, and their embryos are transparent, which enables concurrent imaging and optogenetic control of neuronal network activity and assaying for fear behavior.

 

In this project, we are characterizing the olfactory sensory neurons and odorant receptors that receive the alarm substance stimulus and the patterns of activity that they elicit in the olfactory bulb, the first relay in olfactory sensory processing, as well as in other brain regions. This information will provide the foundation for elucidating the higher order neural circuitry subserving an innate fear behavior. The intersection of a simple, tractable model vertebrate organism; a robust and innate sensory-guided behavior; and the ability to visualize and manipulate neuronal activity in vivo using optogenetics presents an exceptional opportunity to dissect a neural circuit spanning sensation through behavior.



The Ngai Lab BRAIN Initiative Project: Classification of Cortical Neurons by Single Cell Transcriptomics

A major goal of neuroscience is to understand how circuits of neurons and non-neuronal cells process sensory information, generate movement, and subserve memory, emotion and cognition. Elucidating the properties of neural circuits requires an understanding of the cell types that comprise these circuits and their roles in processing and integrating information. However, since the description of diverse neuronal cell types over a century ago by Ramon y Cajal, we have barely scratched the surface of understanding the diversity of cell types in the brain and how each individual cell type contributes to nervous system function. Current approaches for classifying neurons rely upon features including the differential expression of small numbers of genes, cell morphology, anatomical location, physiology, and connectivity – important descriptive properties that nonetheless are insufficient to fully describe or predict the vast number of different cell types that comprise the mammalian brain. This NIH-supported BRAIN Initiative project aims to provide a suite of technologies for identifying and classifying the diverse cell types in the mammalian nervous system. We are developing our method using layer 5 pyramidal cells from mouse somatosensory cortex as a model system.

First, we will exploit the latest developments in DNA sequencing technologies to characterize gene expression profiles on single layer 5 neurons at high throughput. This information will be used to classify individual cells based on their transcriptome “fingerprints.”

Second, genes found to define newly discovered neuronal subtypes will be used to gain genetic access to these cells using Cas9/CRISPR-mediated genome engineering to create transgenic reporter lines. Development of this technology promises to open a pipeline for the rapid generation of multigenic mouse reporter strains in which specific neuronal subtypes are uniquely labeled by combinations of tagged genes.

Third, we will use these genetically engineered mice to confirm that our taxonomy represents distinct functional properties of the classified neurons.

Our approach can ultimately be scaled up to generate a complete census of cell types in the brain, a critically needed resource for dissecting nervous system function with modern investigative tools.

The Ngai Lab BRAIN Initiative project represents a multidisciplinary collaboration between 10 research groups at UC Berkeley working in the following areas:



Genomics

·       John Ngai – PI (MCB)

Neurobiology

·       Hillel Adesnik (MCB)

·       Helen Bateup (MCB)

·       Dan Feldman (MCB)

Genome Engineering and Mouse Transgenesis

·       Jennifer Doudna (MCB, Chemistry, HHMI, LBNL)

·       Dirk Hockemeyer (MCB)

·       Russell Vance (MCB, HHMI)

Statistics and Bioinformatics

·       Sandrine Dudoit (Biostatistics)

·       Elizabeth Purdom (Statistics)

·       Nir Yosef (Computer Science)