Linda J. Richards - US grants

DESCRIPTION (Adapted from the applicant's abstract): This application proposes to examine the role that three glial cell populations play in the development of the corpus callosum. One of these populations form the glial sling, a group of cells that underlie the corpus callosum forming a "bridge" between the two hemispheres. Two newly discovered populations are the glial wedge, a group of cells near the midline that are proposed to deflect cortical axons medially and the indusium griseum, a group of glial cells located dorsal to the callosum. The three specific aims proposed in this new application are as follows: (1) characterize the role Emx1 plays in regulating the expression of guidance molecules derived from the glial sling; (2) determine whether two other midline glial populations are also important for midline guidance of cortical axons; and (3) examine the development of midline glial cells in mouse mutants exhibiting an acallosal phenotype i.e. the netrin-1 and DCC mutant mice.

DESCRIPTION (provided by applicant): The glial sling was first described by Silver and colleagues in the early 1980's as a population of glioblast cells that arise in the medial aspect of the lateral ventricles. These cells migrate toward the midline during embryogenesis and form tight junctions, creating a sling-like structure that spans the two cerebral hemispheres. When the sling is severed the corpus callosum does not form; in congenitally acallosal mice or in marsupials (which lack a corpus callosum), there is no sling. These observations led to the hypothesis that the sling is required for the corpus callosum to form by guiding callosal axons across the midline. Surprisingly, we have recently discovered that the majority of glial sling cells label with neuronal rather than glial markers (see preliminary data). Preliminary electrophysiological experiments indicate that sling cells have excitable membranes and fire action potentials in response to long depolarizing pulses via whole-cell current clamp. Sling cells from postnatal animals display spontaneous action potentials that are reversibly blocked by TTX. Taken together, these new findings indicate that sling cells are neurons, not glia.Previous descriptive studies found that sling cells migrate from the medial subventricular zone to the midline between E15 and E17 in mice and that the sling disappears early postnatally. Once the sling cells reach the midline, it was proposed that they die and form a small cavity called the cavum septum pellucidum. However 11JNEL labeling shows that only a few cells undergo cell death at E17 and E18. Since the sling continues to be generated until P2 (see preliminary data) the absence of the sling by P5 may be due to additional (or other) mechanisms than cell death, such as cell migration away from the sling area. Preliminary experiments in organotypic slices and adenoviral labeling in vivo suggest that sling cells continue migrating past the midline to other areas of the brain. In aim 1 we extend these findings to determine where sling cells migrate in the developing brain. The sling is continuous with the SVZ of the cortex and may represent a previously unknown population of tangentially or contralaterally migrating cortical neurons. In aim 2 we investigate whether sling cells arise only at the cortico-septal boundary from a specialized sling progenitor population, or whether their progenitors are dispersed in more lateral regions of the SVZ. Using a recombinant retroviral library we determine whether sling cells are clonally related to cells of the cortical plate. Prenatally sling neurons are immature, in aim 3 we investigate their differentiation potential and determine if they are committed to a single, or to multiple, neuronal lineages. These experiments redefine the cellular makeup, migration and possible function(s) of the subcallosal sling during cortical development. Development of the sling is disrupted in a number of genetic acallosal mutants that display additional brain abnormalities. Neurons within the sling may function in the development of the brain and defects in their development may underlie some of these disorders

DESCRIPTION (provided by applicant): Although over 50 human congenital syndromes are associated with agenesis of the corpus callosum (ACC), we know very little about why ACC actually occurs in developing human fetuses. Part of the reason for this is that we lack fundamental knowledge about the molecular and developmental requirements for normal callosal formation in humans. In mice we have made significant progress in understanding how the corpus callosum forms. My laboratory has identified several midline glial structures that regulate callosal axon pathfinding as well as some of the genes that are expressed by these structures. We have also been analyzing a number of different mouse mutant strains that phenotypically display ACC and have determined that these genes may play specific and vital roles in callosal formation. This proposal is specifically designed to move our scientific knowledge base in animal models to humans. Using this mechanism of "Exploratory grants in pediatric brain disorders: Integrating the science" we have built a team of professionals with basic science and clinical expertise in midline brain development. Data from experiments described here will form the basis of a fundamental infrastructure of knowledge between the laboratory and clinical settings. This approach is essential to moving this field forward toward an understanding of the basis and treatment of ACC. In aim 1 we determine if midline glial populations and the chemorepellent molecule Slit2, that are required for callosal formation in mice, are present (and therefore possibly act in an analogous way) in human fetal brains. In aim 2 we investigate the expression of a number of genes that cause ACC in mouse to determine if they are expressed at developmentally relevant times and locations in human fetal tissue. This data will enable us to evaluate the potential of these genes as candidates that may be mutated in human cases of ACC. Finally we examine the development of a structure called the commissural plate, that may underlie the formation of all forebrain midline commissures. The commissural plate has been described in human development but it is not known whether its proper formation is required for commissure formation and whether it expresses guidance factors for commissural axons. In aim 3 we address these issues by first analyzing the formation of the commissural plate in human fetal brains using diffusion tensor imaging and then address whether the same structures form in mice where we can more easily study its development. These experiments are the first to address the molecular and genetic basis of ACC in humans and are based on extensive work in mice. Our goal is to determine how ACC occurs in humans and what factors are common amongst the numerous congenital syndromes in which ACC and other commissural defects occur.

Project Summary/Abstract Brain function requires coordinated activation of specific networks engaged in systems that process information in localised and distributed manners. In order to develop such specific networks, the brain engages groups of neurons that fire together in ensembles that can be observed with calcium imaging. Patterns of spontaneous activity in the cerebral cortex are thought to enable the formation of circuits specialised for processing different types of sensory information. How the brain first switches on activity across areas is unknown. I propose to investigate exactly how and when in fetal life these patterns first occur in vivo, what regulates their development, and how they shape neural circuits and later brain function. A major barrier to addressing this question has been that patterns of activity such as patchwork-type activity in S1 and travelling waves in V1 are present at birth in rodents making it difficult to study this question in vivo as the brain apparently switches on before birth. To address this, I propose to apply modern scientific tools and technologies to an Australian marsupial mammal: the fat-tailed dunnart (S. crassicaudata; Dasyuridae), thereby developing a new approach for investigating brain development. Dunnarts are small (adults weigh ~15g), carnivorous animals whose pups (joeys) are born at an equivalent stage of development to embryonic day 10 in mouse or seven-week gestation in humans, and therefore most of their brain development occurs as they develop inside their mother's pouch. Despite this more primitive developmental phase, dunnarts have a six-layered cerebral cortex which is similar to a mouse but with advantageous exceptions such as a more advanced binocular visual system. Dunnarts are also able to solve complex configurable problems and learn quickly. To ensure feasibility of this project, I provide evidence that we can use targeted electroporation to introduce sensitive calcium indicators such as GCaMP6S into the cortex. In preliminary experiments we find that patchwork-type activity in S1 and traveling waves in V1 are evolutionarily conserved in dunnarts, motivating this new direction of my research to understand the development and function of these patterns of spontaneous activity. Having access to study the entire genesis and development of these patterns enables longitudinal studies that can link cells, circuits and behavior/function. The creation of longitudinal imaging capabilities bridging micro/meso/macro scales as well as awake behavior across the lifespan will be required in order to identify which neuronal cell types initiate spontaneous synchronous activity and whether these activity patterns are instructive in forming functionally-specific circuits. I will also explore how spontaneous activity in the cortex evolves throughout life as circuits begin to function to mediate sensory experience and behavioural reactions. I ensembles knowledge propose that by understanding the fundamental processes r equired to build of patterned activity in the brain and how these affect behavior, this work will advance our of the neural basis of mental experience.