Stuart Tobet, PhD
Research Interests -- Determination of Cell Positions in the Developing Neuroendocrine Brain
The long-term goal of my research is to determine cellular and molecular events underlying the differentiation of regions of the brain that underlie neuroendocrine function. Neuroendocrine structures are important because they regulate behavior and hoemostasis and are susceptible to a variety of diseases or syndromes such as Kallmann's, Prader-Willi, or Rubenstein-Taybi. I began my career by examining the long-term consequences of early hormone action on sexual behaviors, reproductive physiology, and hypothalamic structure. I proceeded to focus on molecular actions of gonadal steroids during critical periods of hormone action. We are now determining how multiple signals affect migration and cell position in the developing nervous system.
Cellular organization and differentiation may follow several courses leading to primarily layered (e.g., cerebral or cerebellar cortices) or nuclear (cell groupings within the thalamus and hypothalamus) structures. We concentrate on the formation of nuclear structures using the ventromedial and paraventricular nuclei of the hypothalamus (VMH and PVN) as model systems. The VMH and PVN are part of complex neural circuitries that regulate homeostatic, neuroendocrine, and behavioral functions. They migrate to specific positions within the respective nuclei characterized by neurochemical environment, phenotype of neighboring cells, and the pattern of anatomical connections. Our model system allows us to follow the formation of nuclei in vitro (video microscopy) and is easily accessible to manipulation. Our discovery of a unique relationship of the neurotransmitter GABA to the developing VMH, and more recently the PVN, has led to hypotheses that are being tested directly through pharmacological manipulations and selecting transgenic mouse models. In particular, we are taking advantage of two lines of gene-disrupted mice. In one line, a single gene-deletion (the nuclear orphan receptor, steroidogenic factor-1 or SF-1) leads to failure of VMH formation. In the other, the R1 subunit of the GABAB receptor is disrupted and GABAB receptors are functionally impaired. The unique targeting of the SF-1 gene to the VMH also leads to our efforts to examine transgenic mice in which green fluorescent protein (GFP) expression is driven by the SF-1 gene promoter and another line of mice in which GFP expression is driven by the NPY promoter (see images).
We are also studying GABAergic mechanisms in the development of neurons that synthesize gonadotropin-releasing hormone (GnRH). During embryonic development, neurons containing GnRH migrate from the nasal compartment, across the cribriform plate, into the brain to reside in the basal forebrain. Over their early migration, we have found GABA either in neighboring cells or within GnRH neurons in mice, rats, humans and lamprey. Using pharmacology and a unique in vitro slice paradigm that keeps the relationship of the head and brain intact, we are testing mechanisms of migration directly. Here again, we are taking advantage of lines of transgenic and gene-disrupted mice. In particular, we are examining GnRH neuron migration in mice deficient in the netrin-1 receptor, deleted in colon cancer (DCC). In addition, we are using live video microscopy to examine the migration of GnRH neurons that are revealed by GFP expression driven by the GnRH gene promoter. We are characterizing intracellular, extracellular, and cell surface molecules as they relate to this instance of neuronal migration from the periphery into the CNS, a unique event.
Finally, we were led to examine the role of gonadal steroids in cell migration when a monoclonal antibody we generated revealed sex differences in antigen expression in radial glia in the hypothalamus of perinatal rats. We further discovered a transient radial glial scaffold that reaches from the lateral ventricles across the anterior commissure through the sexually dimorphic preoptic/anterior hypothalamus (POAA/AH) to the pial surface at its base. We are exploring which cells might utilize this unique radial glial pathway using immunocytochemical and live video microscopy techniques. We are particularly focused on following the migratory behavior of cells that might contribute to sex differences in structure or function. Our live video microscopy allowed us to discover the first evidence of sex differences in neuronal migration. Now, it is providing for our examination of hypotheses of the mechanism(s) of sex differences. By focusing our efforts primarily in murine models, we combine the utility of in vitro work and pharmacology with the power of mouse genetics. Thus we are taking advantage of different transgenic mice or mice in which selected genes have been disrupted (e.g., SF-1 mentioned above, and others) to explore the importance of specific steroid hormone systems in the POA/AH.
Knoll JG, Wolfe CA, Tobet SA. 2007. Estrogen modulates neuronal movements within the developing preoptic area-anterior hypothalamus. Eur J Neurosci 26:1091-1099.
McClellan KM, Calver AR, Tobet SA. 2008. GABAB Receptors role in cell migration and positioning within the ventromedial nucleus of the hypothalamus. Neuroscience 151:1119-1131.
Tobet S, Knoll JG, Hartshorn C, Aurand E, Stratton M, Kumar P, Searcy B, McClellan K. 2009. Brain sex differences and hormone influences: A moving experience? J Neuroendocrinol 21:387-392.
McClellan KM, Stratton MS, Tobet SA. 2010. Roles for gamma-aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus. J Comp Neurol 518:2710-2728.
Stratton MS, Searcy BT, Tobet SA. 2011. GABA regulates corticotropin releasing hormone levels in the paraventricular nucleus of the hypothalamus in newborn mice. Physiol Behav 104:327-333.
Majdic G, Tobet S. 2011. Cooperation of sex chromosomal genes and endocrine influences for hypothalamic sexual differentiation. Front Neuroendocrinol 32:137-145.
Lynn NS, Tobet S, Henry CS, Dandy DS. 2012. Mapping spatiotemporal molecular distributions using a microfluidic array. Anal Chem. 84:1360-6.
Frahm KA, Schow MJ, Tobet SA. 2012. The vasculature within the paraventricular nucleus of the hypothalamus in mice varies as a function of development, subnuclear location, and GABA signaling. Horm Metab Res. 44:619-24.