Postdoctoral Position Available

Daniel Kilpatrick, Ph.D.

Academic Role: Associate Professor

Faculty Appointment(s) In:
   Physiology

Other Affiliation(s):
   Cell Biology
   Program in Neuroscience

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 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.  These transcriptional mechanisms also 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 ).  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 recently 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.

We also found that NFI proteins are highly enriched in CGNs relative to other neurons (W. Wang et al, J Biol. Chem. 2004), suggesting a potentially larger role for this family in CGN post-mitotic differentiation.  Our more recent studies have confirmed this (W. Wang et al, J. Neurosci. 2007). We found that NFI family members are essential for proper formation of parallel fibers, for migration of CGNs from the PMZ to the IGL and for dendrite formation, both in culture and in vivo.  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).  We showed that these two cell adhesion molecules regulated 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. Interestingly, we have now found that the NFI family also controls a third cell adhesion molecule, Tag-1/contactin-2, which has a critical role in axon-axon interactions and is highly expressed during parallel fiber formation within the PMZ. Thus, cell adhesion molecules are critical downstream targets of the NFI family in differentiating CGNs. Additional NFI gene targets are now being defined.

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. These studies will have important implications for  neurodevelopmental disorders in which specific maturation events and their timing are altered within the cerebellum and elsewhere in the CNS.

Selected Recent Publications:

Sartini, B.L., Wang, H., Wang, W. and Kilpatrick, D.L. “Pre-Messenger RNA Cleavage Factor I (CFIm): Potential Role in Male Germ Cell-Specific Alternative Polyadenylation.” Biol. Reprod. 78(3):472-82 (2008).

Wang, W., Mullikin-Kilpatrick, D., Crandall, J. E., Gronostajski, R. M., Litwack, E.D. and  Kilpatrick, D.L. “Nuclear Factor I Controls Multiple Phases of Neuronal Development via Regulation of Cell Adhesion Molecules.”   J. Neurosci. 27, 6115-6127 (2007).

Wang, H., Sartini, B.L., Millette, C.F. and Kilpatrick, D.L. “A Developmental Switch in Transcription Factor Isoforms During Spermatogenesis Controlled by Alternative 3’-end Formation.”  Biol. Reprod. 75: 318-323 (2006).

Wang, W., Qiang, Q. Smith, F.I. and Kilpatrick, D.L. "A Self-Inactivating Lentiviruses: Versatile Vectors for Transduction of Cerebellar Granule Neurons and Their Progenitors."  J. Neurosci. Methods 149: 144-153 (2005).

Wang H., San Agustin, J.T., Witman G.B. and Kilpatrick, D.L. "A Novel Role For An SREBP In Directing Spermatogenic Cell-Specific Gene Expression."  Molec. Cell. Biol. 24:10681‑10688 (2004).

Wang W., Stock, R.E., Gronostajski, R.M., Wong, Y.W., Schachner, M. and Kilpatrick, D.L.  "A Role for Nuclear Factor I in the Intrinsic Control of Cerebellar Granule Neuron Gene Expression."  J. Biol. Chem. 279:53491-53497 (2004).

Kim Y.S., Nakanishi G., Oudes A.J., Kim K.H., Wang H., Kilpatrick D.L., Jetten A.M. "ATsp57: a novel gene induced during a specific stage of spermatogenesis."  Biol Reprod. 70:106‑113 (2004).

Rotation Projects

Transcriptional Determinants of Neurogenesis

During neurogenesis, neural progenitor cells in different regions of the nervous system give rise to neurons (and glia) in a process known as terminal differentiation. While all neurons share several common features, each population possesses a unique phenotype related to their specific functions (e.g., dendritic fields, axonal projections and synaptic connections, neurotransmitters and receptors, etc). Further, the onset of this process is initiated once neuronal progenitors exit the cell cycle. That is, progenitor growth arrest activates a series of transcriptional pathways that give rise to not only general neuronal features, but also to neuronal subtype-specific gene expression. We are interested in understanding the transcriptional control of this process at several levels in the context of cerebellar granule neurons (CGNs). We are currently addressing the following questions:

1. How is the decision by neuronal progenitors to stop dividing linked to onset of the terminal differentiation program? In this case, we are identifying those transcription factors that operate at the interface between the growth arrest and differentiation programs in CGNs, whit a specific focus on the E2F family. These proteins play a pivotal role during cell cycle events, and we have recently demonstrated their importance in neuronal differentiation as well. We are now performing functional studies of E2F proteins using viral expression vectors as well as knockout mice to determine their precise roles.



2. Which transcription factors participate in the regulatory cascades leading to the final stage of terminal differentiation, as well as neuronal subtype specificity? Here we are focusing on the late phase of terminal neuronal differentiation, dendritogenesis-synaptogenesis, in CGNs. Little is currently known of how these final, critical events of neurodevelopment are elaborated. To this end, we are identifying and functionally characterizing the key transcriptional players controlling these processes using: a) microarrays to identify candidate transcription factors that are developmentally expressed during late terminal differentiation; b) transgenic promoter analysis and expression cloning of DNA binding proteins to define the factors regulating the GABAA 6 receptor subunit gene, which is highly upregulated during dendrite formation of CGNs. This gene is also expressed exclusively by CGNs, and thus provides insight into transcriptional mechanisms controlling neuronal subtype-specification. Viral expression vectors and in vivo studies will be used to define the specific roles of transcriptional regulators identified using the above approaches.

Laboratory Personnel:

Academic Background

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

Office: S4-139
Phone: 508-856-6274
E-mail: Daniel.Kilpatrick@umassmed.edu
Keywords: Neurobiology, Gene Regulation, Testis Development, Developmental Biology, Transgenic Mice

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Postdoctoral Position Available An NIH-funded postdoctoral position in Molecular Neuroscience is available immediately to study transcriptional programming and chromatin regulation of neurodevelopment and associated disorders. Our lab uses multiple approaches including gene knockouts, BAC transgenics, shrnas, viral expression, microarrays, chromatin IP, ChIP-Seq and related molecular techniques.  Ph.D. with experience in molecular biology is important, and expertise in protein-DNA interactions and/or molecular neuroscience preferred.  Send CV and names of three references to: Dr. Dan Kilpatrick, Department of Physiology and Neuroscience Program, UMASS Medical School, Worcester, MA  e-mail: daniel.kilpatrick@umassmed.edu. The University of Massachusetts Medical School is an Affirmative Action / Equal Opportunity Employer.