Development and Function of the Mouse Vestibular System in the Absence of Gravity Perception.
(In collaboration with Dr. Debra J. Wolgemuth, Ph.D., Columbia University, New York)

Objective

The long-term goal of the proposed studies is to assess the potential effects of the altered environment associated with space flight on specific aspects of neural development in mammals. The space flight environment involves several unique influences including microgravity, a physical factor rarely experienced by species on the Earth. The hypothesis to be tested in this proposal is that the absence of gravity perception, such as would occur in space, will affect the development and function of the vestibular and central nervous systems. Further, we postulate that these effects will be more significant at specific stages of post-natal development of the animal. We also propose molecular genetic approaches that will provide important information as to the heirarchy of genetic control during the development and subsequent function of the vestibular system. While the ideal setting for examining the effects of these parameters is flight itself, the opportunities for such experiments are severely limited. Ground-based studies are therefore crucial as a means for evaluating the stages of development most likely to be affected by space flight as well as for identifying genes that are essential for normal vestibular development and vestibular physiology. The recent availability of mutations affecting specifically vestibular function in mice makes this task practically achievable. The tilted (tlt) mutant mouse has been characterized by dysfunction of the gravity receptors. Homozygous tlt/tlt mice specifically lack otoconia, which contain crystals of calcium carbonate that excite the sensory epithelium in the utricular and saccular maculae in response to the change of direction or force of the gravitational field. Consequently, tlt/tlt mice do not have a sense of spatial orientation relative to the force of gravity. The tlt/tlt mutants are a particularly attractive model for the study of vestibular function since unlike other mouse mutations affecting the vestibular system, the primary defect in the tilted mouse is limited to the most proximal part of the receptors of the vestibular system. There are no abnormal phenotypes in other organ systems.

Specific Aims

The goal of the proposed studies is to investigate the postnatal neural development of the tlt/tlt mutant mouse. We wish to (a) assess immediate and delayed effects on the vestibular system with particular focus on characterization of primarily affected periods of vestibular morphogenesis, and (b) to identify downstream genetic pathways that are altered in the CNS of the tlt/tlt mutant mouse, which lacks gravity receptor input. The specific aims for the three-year period of investigation are:

1.  To characterize the postnatal morphogenesis of the vestibular system in the tlt/tlt mutant mouse, with particular focus on the vestibular ganglion and the vestibular nuclei in the brain stem. The proposed studies will involve detailed morphometric analysis of isolated vestibular ganglia and brain tissue from the mutant animals at different stages of postnatal development. A second aspect of assessment of the differences between normal and tlt/tlt mutant mice will address specifically any changes in apoptotic cell death during development of the vestibular system in the absence of perception of the gravity stimulus. [Years 1-2]

2.  To begin to understand the molecular basis for perception of and response to gravity in the vestibular system, we will examine the expression of potentially affected genes, by in situ hybridization or immunohistochemistry, in the tlt/tlt nervous system during post-natal development. The candidate genes have been selected by virtue of the fact that mutational analysis has shown that they can have profound affects on the development or function of the vestibular system. They include BDNF, NT-3, and the Trk B and Trk C receptors. [Years 1-2]

3.  To identify other genes involved in vestibular development and function, we will perform differential cloning strategies for genes whose expression is changed in the tlt/tlt mutant versus normal vestibular system. The approaches will utilize representational difference analysis (RDA) of mRNAs isolated from dissected brain structures, including the vestibular ganglion and vestibular nuclei of the brain stem. The initial screen will focus on the stage of post-natal development when the first morphometric changes are observed in our analysis in Specific Aim 1.

Significance

A pressing issue in space biology is how and in what manner normal developmental mechanisms will be affected in the altered environment of space. Postnatal neural development relies heavily on an appropriate sensory environment and is very sensitive to altered environmental stimulation and stresses. The environment of space could therefore affect neural development and result in biologically significant changes in the animal's morpho-physiology and behavior. Preservation of developmental programs represents a critical issue for the long-term survival of all species. Understanding the mechanisms by which gravity influences normal neurological development is a critical issue of space developmental biology, evolutionary biology, and organismic biology. The ability to sense gravity was developed early in evolution. Critical for the ability to orient in the space and to maintain equilibrium, the sense of gravity has been crucial for species survival. Correspondingly, the vestibular system is one of the oldest and most highly conserved parts of the sensory systems (Romer, 1964). Among the different environmental stimuli, gravity is one of the first forces that a newly born animal faces. Adaptation of organisms to gravity occurs mainly postnatally and includes maturation of the vestibular system, including both the peripheral and central compartments. It is our proposition that the absence of the gravity stimulus, as would occur in the environment of space, may have significant impact on the normal development of the vestibular system. Investigation of the effects of altered gravitational stimulation on development of the vestibular system will both contribute to a better understanding of the possible immediate and delayed effects of the space flight environment on postnatal neural development. Specifically, current proposal will allow the identification both of periods of vestibular morphogenesis as well as genes affected by lack of the sense of gravity.

Funded by NASA (NAG 2-1345)