Academic Background

B.A., University of California at San Diego,
Revelle College, 1974
Ph.D., Duke University, 1980

Transcriptional Programming of Neuronal Differentiation and its Linkage to CNS Disorders

Post-mitotic maturation of neurons occurs in discrete stages, including migration, axon extension, dendritogenesis and formation of functional synaptic connections. Elaboration of these events requires the expression of specific gene subsets in the appropriate sequence and patterning, and alteration of this sequential expression can disrupt neuronal development. A central and unexplored question is how the precise timing and ordering of such developmental events is coordinated within maturing post-mitotic neurons. We are deciphering this regulatory code since it may have important implications for stem cell therapies of neurodegenerative diseases as well as for a variety of neurodevelopmental disorders. For example, several forms of mental retardation, autism spectrum disorders as well as schizophrenia and epilepsy have been linked to transcriptional dysregulation during development. Further, several of these disorders exhibit temporal delays or alterations in neurodevelopment or in neurologically-based behaviors. Further, these transcriptional mechanisms may be relevant to adult neurogenesis, synaptic plasticity and learning/memory-associated events.

It has become quite clear that the transcriptional differentiation program in neurons is not a linear cascade, but a dynamic, interactive network that changes over time. The present challenge is therefore to elucidate the key transcription factors that comprise such networks and how their actions are integrated into a coherent program that controls both neuronal specification and sequential expression of relevant genes. A related question is the nature of the downstream targets that mediate the actions of these trans-regulators as part of an overarching differentiation program.

Figure 1. Differentiation of granule neurons within the developing postmitotic cerebellum. oEGL, outer external germinal layer (site of granule neuron progenitor proliferation). PMZ, pre-migratory zone (where immature granule neurons extend axons). ML, molecular layer (site of parallel fiber/Purkinje neuron synapsis). PL, Purkinje cell layer. IGL, internal granule cell layer (final destination of granule neurons following migration from PMZ; site of dendrite formation and terminal  maturation). Examples of stage-specific gene expression are shown to right.

Nuclear Factor I:   A Central Regulator of Neuronal Development

Cerebellar granule neurons (CGNs) undergo a well-defined sequential program of differentiation that serves as an excellent model for various aspects of neuronal development (Figure 1) (see also DL Kilpatrick et al., Cerebellum 2010). In the postnatal cerebellum, granule neuron progenitors proliferate in the outer portion of the external germinal layer (oEGL). Immature CGNs take up residence within the premigratory zone (PMZ) where they elaborate bipolar axons (parallel fibers) along which their cell bodies migrate tangentially. CGNs then extend a third, radial process and migrate radially along Bergmann glia to form the internal granule cell layer (IGL). Post-migratory CGNs complete their differentiation in the IGL by forming dendrites and synaptic connections with mossy fibers and Golgi type II neurons. As part of this program, numerous genes are expressed in distinct temporal patterns in order to promote these different maturation steps (Figure 1).

We have begun to explore the underlying transcriptional mechanisms responsible for the regulation of these different phases of CGN development and the key downstream targets that mediate these events. Members of the Nuclear Factor I (NFI) family (NFIA, NFIB, and NFIX) have been directly implicated in nervous system development, although their specific functions and gene targets have not been defined previously. Using a combination of culture and gene knockout approaches, we previously found that NFI proteins have a primary role in regulating the Gabra6 gene in maturing CGNs (W. Wang et al, J Biol. Chem. 2004). For example, NFIA knockout mice have greatly reduced Gabra6 expression in the cerebellum. Transcription of the Gabra6 gene in vivo does not occur until CGNs finish their migration and initiate dendritogenesis in the internal granule cell layer (Figure 1). Thus, NFI proteins are critical for expression of a gene that is expressed very late in CGN maturation.

Subsequent studies (W. Wang et al, J. Neurosci. 2007) found that NFI family members are essential for multiple stages of CGN development: formation of parallel fibers, radial migration of CGNs from the PMZ to the IGL and dendrite formation. Thus, NFI proteins have a global impact on the maturation of CGNs throughout their post-mitotic development. A key question arising from this is how a single family of transcription factors is able to direct the completion of sequential phases of CGN differentiation. Further findings provided at least one answer to this question. We found that the actions of NFI proteins are mediated through the direct regulation of cell adhesion molecules, including Ephrin B1 and N cadherin (Cdh2) (W. Wang et al, J. Neurosci. 2007). These two cell adhesion molecules were shown to regulate CGN axon formation, migration and dendritogenesis. Further, they are expressed throughout the CGN differentiation program, thus providing a means for NFI regulation of diverse maturation phases. More recently, we found that the NFI family also controls a third cell adhesion molecule, Tag-1/contactin-2, which is highly expressed during parallel fiber formation within the PMZ (W. Wang et al., J. Neurosci. Res. 2010). Tag-1 was subsequently shown to play an important role in axon extension and cell migration by maturing CGNs (W. Wang et al., Cell. Molec. Neurobiol. 2010). Thus, cell adhesion molecules are critical downstream targets of the NFI family in differentiating CGNs. Additional NFI gene targets are now being defined.

Transcriptional Timing Mechanisms in Developing Neurons

Gabra6, Tag-1, Cdh2 and Ephrin B1 are each expressed in CGNs in distinct temporal patterns (Figure 1 and other data), and current findings indicate a central role for NFI in this differential temporal patterning. How does NFI regulate the temporal expression of multiple genes expressed with distinct timing patterns? This is likely to involve multiple mechanisms, including NFI interactions with other trans-factors. Defining these mechanisms is a central focus of our current work. Very recently (W. Wang et al., Molecular Biology of the Cell, 2011) we identified an interesting mechanism for temporal control of the Gabra6 gene, which is expressed very late in CGN development. It was found that the timing of Gabra6 expression is linked to delayed onset of NFI occupancy of its binding site in the Gabra6 promoter, and that the trans-repressor REST controls the temporal onset of NFI binding and Gabra6 gene activation. Overall, defining temporal mechanisms in maturing neurons may have important implications for neurodevelopmental disorders in which specific maturation events and their timing are altered within the cerebellum and elsewhere in the CNS.