Our research is aimed at the identification of molecular mechanisms that control development and dysfunction of the mammalian cerebellum. We have chosen to study the cerebellum because it is a complex and highly stereotyped structure in which major pattern formation and functional organization occur postnatally. During the past century, classical neuroanatomical studies have very precisely described the complex events that must occur for formation of this brain structure, providing what might be considered as the most detailed information available concerning the development of a given brain region. An extensive literature has documented the importance of cell-cell interactions in the development and maintenance of normal cerebellar architecture, although the mechanisms mediating these interactions remain unknown. Identification of these mechanisms, and analysis of their functions throughout the central nervous system (CNS), are major priorities of the Heintz laboratory.

One strategy for investigation of molecular mechanisms controlling cerebellar development is the isolation and investigation of specific genes that mark critical events in its formation. During the past several years, we have completed a comprehensive screen to identify cDNAs that are precisely regulated during the specification and differentiation of particular cerebellar neurons and glia. These molecules provide a direct avenue for the investigation of cell:cell signaling events that play a critical role in this process. Furthermore, in most cases the strict temporal and spatial regulation of these genes portends a very specific functional role for their products in cerebellar development. A second approach toward understanding molecular events that are crucial in cerebellar formation and function is provided by the analysis of neurologic mutant mouse strains in which specific cerebellar neurons degenerate as a consequence of the disease. We have utilized both of these avenues to begin to dissect complex developmental pathways that play a role in development of the brain.

Granule Cell Specification in the Developing Brain. The generation of specific neuronal cell types in the developing brain is a critical issue. Although very little is known concerning molecules that control cell specification in the mammalian central nervous system, precedent from other organisms and from the development of other mammalian organs has established the role of cell type specific transcription factors in these processes. During the past year, we have discovered a mammalian zinc finger transcription factor (Ru49) with characteristics that suggest a role for this protein in the establishment of granule cell lineages in the mammalian brain. We have determined that Ru49 possesses a novel DNA binding specificity, that functional Ru49 is present in the developing nervous system, and that it is expressed in cerebellar granule cells from their birth at the rhombic lip throughout their complex differentiation program, remaining evident in mature granule cells in the adult cerebellum. This factor is also expressed in the olfactory and dentate gyrus granule cell lineages, labeling both their earliest precursors within the neural tube and remaining expressed in the fully mature neurons. These results suggest a role for Ru49 in specification of these three cell types in the developing brain.

Neural Differentiation in the Developing Cerebellum. The differentiation of specific cell types in the cerebellum requires complex molecular subprograms that provide distinct functions for differentiating cells. Many of these functions are generally important for neuronal differentiation, being shared by different cell types to accomplish a given task. One example of such a subroutine is glial guided neuronal migration, which is necessary for the translocation of many different neurons from their place of birth in the neural tube to the eventual sites of their maturation in the developing brain. During the past year, studies in our laboratory and in collaboration with Dr. Mary E. Hatten of The Rockefeller University, have yielded important insights into this problem. Previous studies in our laboratories have established that the brain lipid binding protein (BLBP) is critical for the differentiation of primary glial cells in vitro, and that this protein is produced in radial glial cells only during the period of neuronal migration. During the past year, we have identified a transcriptional regulatory element that is critical for the regulation of BLBP transcription in radial cells in vivo, and have shown that this same element can program expression of the BLBP gene in glial cells in primary culture only if differentiating neurons are present. These results suggest that the dynamic regulation of BLBP expression in vivo, and hence radial glial function, is a consequence of neuron:glial interactions that maintain glial differentiation. Additional studies have led to the cloning of the mouse astrotactin gene, to its identification as a novel neuron specific signaling molecule, and to the demonstration that it is involved in migration and assembly of differentiating neurons into cortical structures of the mammalian brain. This work, and additional studies of other molecules important for cellular differentiation in the developing cerebellum, remain an important priority in the laboratory. (These studies are partially supported by the National Institutes of Health.)

Cerebellar Dysfunction: Positional Cloning of Murine Neurologic Mutant Genes. A second area of investigation in the Heintz laboratory is the identification of genes responsible for mouse neurologic diseases. The phenotypes of strains carrying these mutations identify the affected genes as essential for either normal cerebellar development, or for its maintenance in mature animals. For example, the Lurcher (Lc) mutation results in degeneration and death of essentially all cerebellar Purkinje cells commencing ~10 days after birth. Detailed analysis of the molecular events underlying Purkinje cell death in Lc mice strongly suggests that these cells die by the process of apoptosis. It is our hope that identification of the Lc mutation will provide fundamental insights into the ectopic activation of programmed cell death in inherited neurodegenerative diseases. During the past year, we have completed detailed genetic and physical mapping of the Lc mutation, and have constructed a YAC contig spanning this locus. A single YAC clone containing the Lc gene and the four closest recombination break points spanning it has been isolated, and is presently being utilized to select cerebellar cDNAs as possible candidates for the Lc gene. Similar studies of the meander tail (in collaboration with Dr. Colin Fletcher, National Cancer Institute) and nervous mutations are also being pursued in the Heintz laboratory.

Control of Gene Expression during the Cell Cycle. Dr. Heintz's laboratory has established that transcriptional induction of histone gene expression involves coordinate activation of a set of transcription factors (Oct1, H1TF2, H1TF1,H4TF2) that interact with subtype specific consensus sequences within the histone gene promoters. These sequences, and their cognate DNA binding proteins, are critical for cell cycle regulation of this gene family. Recent studies in the Heintz laboratory have established that the histone gene transcription factors undergo a complex program of phosphorylation during the cell cycle that eventually leads to their inactivation in mitosis. However, no direct modification of these proteins has yet been found that could explain histone gene transcriptional induction at the beginning of S phase.

During the past year, the laboratory has concentrated on identifying proteins that interact with the H2b transcription factor Oct1 and the H1 transcription factor H1TF2. Although these two transcription factors are both involved in cell cycle regulation, their primary structure and biochemical properties are entirely different. Present efforts focus on biochemical characterization of proteins associated with these transcription factors in vitro, and immunoprecipitation studies of factors interacting with Oct1 and H1TF2 in vivo, in efforts to identify a common cofactor that might participate in coordinate control of S phase specific transcription. The discovery of a mechanism for transcriptional regulation of histone gene expression during S phase can provide important insights into control of progression through the mammalian cell cycle.