Pasko Rakic, MD, PhD

The long standing objective of research in this laboratory is to understand the cellular events and molecular mechanisms that govern development of the mammalian central nervous system. One line of investigation focuses on the fundamental issue of the regulation of cell proliferation and death (apoptosis) that determine the number of neurons allocated to the building of the cerebral cortex. The other series of studies concern molecular mechanisms involved in neuronal migration including cell-cell recognition, neuron glia-interaction and nuclear translocation.

Specialized Terms: Central nervous system development

Research in this laboratory is focused on developmental neurobiology, more specifically on the mechanism of neuronal proliferation, migration, programmed cell death, axonal guidance, and patterns of synaptic connectivity and their plasticity during development of the central nervous system. One line of investigation focuses on the interaction between neuronal and glial cells during neuronal migration, the effects of various epigenetic factors on the development of structural, molecular and functional cell phenotypes, and their segregation into topographic maps.

Other research concerns the differentiation, synaptogenesis, and emergence of transmitters and their receptors in laminated structures (cerebellum, hippocampus, neocortex). Special emphasis has been on normal and experimentally altered development of the visual system following selective destruction of visual centers and/or pathways in developing primates. A battery of the most advanced methods are used with the hope of gaining insight into epigenetic sequences and the cellular and molecular mechanisms that regulate the development and evolution of the primate brain.The cerebral cortex provides the biological substrate for human cognitive capacity and is, arguably, the part of the brain that distinguishes us from other species. Therefore, understanding the evolution and development of this complex structure is central to our understanding of human intelligence, creativity and disorders of the highest cognitive functions. However, in our enthusiasm for universality of biological principles and a conservation of the genomic sequence, we sometimes forget that the heterogeneity among species is higher than expected from their genomic similarity. Even a small difference in the timing, sequence and level of gene expression may influence the phenotype in a major way. It seems obvious that one cannot understand specific human characteristics by looking only at a mouse model that does not possess these traits or have traits that have been lost.

We have new evidence that the species-specific size and characteristics of the cerebral neocortex are already detectable in the embryonic human forebrain before the onset of cortico-neurogenesis, but these early stages are not understood. Thirty-five years ago, the Boulder Committee recommended a blueprint and nomenclature for the development of the mammalian central nervous system, based on the model proposed by the applicant of this proposed grant. This model has been reproduced directly or in a modified form, in virtually every textbook of neuroembryology and the neurosciences, in general. However, the introduction of advanced methods and new concepts in developmental biology now necessitates an update of this basic schema.

The primary objective of the proposed research is to enhance our understanding of the molecular and cellular mechanisms underlying neuronal production, migration and positioning during early development of the cerebral cortex directly in humans. We propose to focus on the initial specification and cascade processes that establish the morphological and functional diversity of neurogenesis in the embryonic human telencephalon. We have recently obtained novel and unexpected evidence that the embryonic human telencephalic primordium, before the onset of local corticogenesis, contains a hitherto unrecognized transient population of neuronal cells. These "predecessor" cortical stem cells (new word!), are situated above the proliferative ventricular zone of the human telencephalon at the 4th week of gestation. We propose a radical idea that these neuronal stem cells are not homogeneous, but rather are already different amongst themselves. Furthermore, we speculate that these cells might initiate the cascade of developmental events leading to the formation of the cerebral cortex, which commences in human only about two weeks later. I am proposing that we try not only to characterize these new types of neural stem cells, but also to isolate and immortalize them with the most advanced methods.

Composite analyses of the gene expression patterns that we are going to perform will allow us to characterize the molecularly distinct progenitor domains in the forebrain primordium and to elucidate the genetic interactions underlying the early regionalization of the telencephalon directly in humans. We have unpublished evidence, which if elaborated, could alter one of the main concepts in the field of developmental neurobiology; namely, that the first neurons in the primordium of the human cerebral cortex (preplate zone) are generated outside of the developing cortex. To confirm these results, we are going to first perform modern cell-tracing experiments in slice culture preparations as well as immunohistochemical analysis to uncover the pattern of migration, connectivity and the role(s) of the pioneer neurons in the human telencephalon.

As a second step, dividing cells situated on the surface of the ex vivo human telencephalic vesicle obtained by legal abortion will be transiently transfected with a plasmid expressing fluorescent marker (GFP) under the control of the retroviral promoter. We will make shallow injections under the pial surface and after a brief recovery period, cells will be dissociated, single GFP expressing cells isolated, expanded with appropriate growth factors and then cryopreserved as cell aggregates. To produce stable clonal cell lines, cells will be infected with a retroviral vector expressing the v-myc fusion protein, propagated and characterized.

The project is designed to combine the expertise and unique experience in human brain development in my group at Yale, USA (including A. Ayoub and K. Hashimoto-Torii), C. Blakemore’s group at Oxford, UK (including I. Bystron and S. Lindsay) and V. Otellin’s group (Institute of Experimental Medicine, St. Petersburg, Russia). My laboratory has applied several new approaches in human brain tissue that involve a combination of organotypic culture and viral transduction to study the molecular mechanisms of cortical development (2); the Oxford group will provide expertise in the characterization of human neuronal cell lines (e.g. Bystron at al, 2005) and in vivo transplantation assays, and the St. Petersburg group has access to fresh embryonic brain tissue from legally performed interruption of pregnancies.

The emphasis on the early aspects of neuronal production, migration and positioning in human brain development will provide new insight into molecular evolution as well as in the pathogenesis of neuro-psychiatric disorders which affect millions of people of all ages which severely impacts the national economy. This information can only be obtained by studying early human forebrain neural stem cells.