Overview

           The overarching question that defines the research interests of my laboratory is how cells progressively acquire and
maintain their unique identity during early vertebrate embryonic development.  We wish to understand the continuous interplay
between the cell-cell interactions and gene regulation that mediate the process by which embryonic cells adopt a specific fate.
Of particular interest is the molecular basis of the early, and often continuing, plasticity of this cell fate.  Because of the large
embryos, external fertilization,  easy access to the presumptive nervous system at early stages, and the well developed embryology
and molecular biology,  we employ the amphibian Xenopus laevis as the model organism for our experiments.   Our efforts have
focused on the areas outlined below.

Neural Determination and Patterning

         A particular area of interest in my laboratory is the determination and patterning of the early vertebrate central nervous
system. We are currently focusing on how cells in the developing brain acquire specific neuronal phenotypes; in particular
we are concentrating on neurons that release the neurotransmitters GABA (the most abundant inhibitory neurotransmitter in
the nervous system) and glutamate (the most abundant excitatory neurotransmitter in the nervous system).   In addition to
using existing molecular markers, we have also isolated additional cDNAs (xGAT1, xVGlut1) that identify GABAergic and
glutamatergic neurons.  Our approaches to addressing the question of neurotransmitter phenotype determination include:
the use of a primary cell culture system to analyze the state of specification of presumptive neurons at different developmental
stages and the ability of various growth factors to alter the fate of these cells;  the use of morpholinos, repressor constructs,
and over-expression constructs to perturb gene expression and determine the effects on GABAergic and glutamatergic
phenotype determination; Xenopus transgenesis to dissect the regulation of specific genes in GABAergic and glutamatergic
neurons.
            Our laboratory is also interested in early developmental plasticity in the nervous system. Using the amphibian Xenopus
laevis that is amenable to both tissue manipulations and molecular analysis,  initial work focused on defining the state of
commitment of cells in presumptive neural tissue at various stages of development.  Using several available as well as newly
identified regional molecular markers, we employed an explant system to show that at mid-gastrula stages the presumptive
neural plate retained enormous plasticity in terms of anterior-posterior determination, a plasticity lost by neural plate stages.
The dorsal-ventral axis of the nervous system exhibited a significant but less profound degree of plasticity even at mid-
gastrula stages.  To further analyze this plasticity, we have developed a system using rotation operations in which the
presumptive neural plate from forebrain through anterior spinal cord is rotated 180 degrees and gene expression
subsequently analyzed. When performed at mid-gastrula stages, these embryos form a normal axis. We have identified a
characteristic signature of genes that are expressed in the different regions linking the plasticity with gene expression.
Our goal is to define the molecular mechanisms mediating this plasticity.

The Role of Hypoxia in Neural and Vascular Development

        An equally important research direction is understanding the role of hypoxia in early vertebrate embryogenesis,
particularly the role of  HIF (Hypoxia Inducible Factor) 1-a.  This  highly conserved gene encodes a protein belonging to
the PAS family and serves as a master regulator for an organism's response to low oxygen levels.  We have isolated a
2Kb upstream region of the Xenopus HIF1a gene and, using Xenopus transgenesis, demonstrated that a 173 bp fragment
is able to drive normal expression of the gene in vitro and in vivo.  In addition, our analysis demonstrated that HIF1a is
differentially expressed with elevated mRNA levels in the nervous system and responsive at the transcriptional level to
hypoxic conditions.  Further experiments will delineate the precise elements required for this regulation.   More recently,
a number of studies have indicated that HIF1a and hypoxia may play critical roles during the course of normal embryonic
development. In order to understand the role of low oxygen mircoenvionments and HIF1a within the embryo,  we are
engaged in experiments to correlate hypoxia with HIF1a expression at both the mRNA and protein level using in
vivo transgenic approaches, and to determine the effects of perturbing HIF1a expression during embryogenesis in
both normoxic and hypoxic conditions.
 

Collaborative Projects:  In Vivo Gene Imaging

       While the various in vitro techniques provide valuable information, and the transgenic technology in Xenopus allow
for innovative in vivo approaches, accurate assessments of plasticity, determination and gene function in the
“post-genomic” era will ultimately require successful in vivo gene expression approaches in mammals. Towards this
end we have undertaken a highly interdisciplinary and collaborative project with a team of detector physicists and
computer scientists from the Physics Department and the Jefferson Lab to develop a dual modality SPECT-based
(single photon emission computed tomography) detector system. We currently are able to image Iodine-125 labeled
compounds in living small animals at an approximately 1mm level of resolution. We continue to improve on this system
and are currently experimenting with the use of optical imaging and the addressing the very significant problem of
delivery and clearance of the labeled compounds using optoporation in collaboration with
faculty in the Physics Department.