On June 21 and 22, 1999, the National Institute on Deafness and Other Communication Disorders (NIDCD) and the Life Sciences Division of the National Aeronautics and Space Administration (NASA) co-sponsored a program planning workshop, "Role of Transgenic and Knockout Studies in Understanding Sensory-Motor Performance in Altered Gravitational Performance." The seed that germinated into this workshop was planted by Dr. Frank Sulzman, of NASA, during a meeting of NIDCD and NASA Life Sciences Division senior staff in June 1998. This workshop explored the viability of studying the effects of altered gravitational exposure on sensory-motor function in genetically altered experimental model systems. The planned outcome of the workshop, if research opportunity was demonstrated, is a jointly-sponsored Request for Applications (RFA) or Program Announcement (PA) for exploratory/developmental grant applications to stimulate scientific activity in this area.
In addition to the co-organizers, Drs. Daniel A. Sklare and David L. Tomko, of the NIDCD and NASA, respectively, sixteen scientists from both the intramural research programs of the NIDCD and NASA and the extramural research community participated in the meeting. These scientists represented disciplines that included molecular biology, molecular genetics, developmental neurobiology, auditory and vestibular morphophysiology, gravitational biology and motor control. The meeting was chaired by Dr. Carey D. Balaban, of the University of Pittsburgh.
The Workshop was organized into three sessions: 1) a tutorial session, 2) a scientific presentation session, and 3) a discussion and writing session. The tutorial presentations provided background information to the workshop participants on developmental biology, gravitational biology, acceleration physiology and methods for generating genetically altered animals. There were also presentations describing current NIH and NASA programs and initiatives in some of these areas. The scientific presentations emphasized developmental processes, encompassing such areas as: 1) the role of homeobox genes and growth factors in inner ear development and plasticity, 2) effects of altered gravitational loading on the development of the inner ear gravity receptor, 3) effects of altered gravitational loading on developing and mature neuromuscular systems and 4) molecular mechanisms that determine muscle fiber diversity and hypertrophic responses.
Summary of Tutorial and Scientific Presentations
Recent advances in molecular genetics have greatly enhanced the search for the genetic determinants of complex biobehavioral functions. The generation of mutant and transgenic animal models, involving such species as the mouse, Drosophila, zebrafish and Caenorhabditis elegans (C. elegans), provides powerful tools for elucidation of these functions. By illustration, a large number of zebrafish and mouse mutants with vestibular system involvement have been developed. Most have phenotypes with multiple defects and variable penetrance and expression. Notable exceptions are the tilted and tilted head mouse mutants, having only one identified phenotypic defect, the absence of otoconia, and 100% penetrance. The genetic bases for these mutants have not yet been established. By contrast, targeted deletion of genes such as Math1 (a mouse homolog of Drosophila proneural gene atonal) or sequences encoding BRN 3.1 and BDNF produces congenital sensorineural anomalies of the inner ear. Although the existence of additional anomalies of the nervous system or other organ systems is difficult to exclude, the existing animal models are a valuable resource for understanding molecular bases for normal inner ear development.
The expanded use of mutant and transgenic animals offers many promising tools for investigating molecular bases of balance function and sensorimotor performance. For example, the identification of regulatory sequences of genes expressed specifically in the vestibular system will allow the targeted introduction of foreign genes into the vestibular system of transgenic mice. These genetically-engineered animals could be designed to create specific animal models of human disease. In addition, such studies could facilitate targeted expression of potentially therapeutic genes to the vestibular system epithelium.
The vestibular system is the primary sensor of angular and linear (including gravity) acceleration of the head. However, since all tissues in the body have mass, gravitoinertial acceleration also affects regional blood distribution, pulmonary function, movements of the abdominal viscera within the peritoneal cavity and both intraocular and intracranial pressure. Hence, sensory signals from these other organ systems also contain information about instantaneous linear and angular acceleration of the body. The sum of constant linear accelerations may be termed the gravitoinertial environment. Established chronic manipulations of effects of the gravitoinertial environment on different tissues include hypergravity, microgravity, exposure of fish embryos to simulated “free-fall” in a bioreactor and tail suspension/hindlimb unloading models in mammals. Since these manipulations affect multiple sensory systems, particular attention must be devoted to controls that discriminate vestibular from non-vestibular effects of exposure to altered gravitoinertial environments.
Gravitational loading plays an important role in the development of the gravity-sensing organs, central motor pathways and both the structure and function of the skeletal muscles. Since the behavior of terrestrial animals has evolved under the static (1G) gravitational environment of earth, both the direction and magnitude of gravitational acceleration may be important implicit variables in molecular processes related to development, maturation and aging of the inner ear and sensory-motor performance. The gravitational load also shapes the development and maturation of the extra-vestibular (e.g., proprioceptive) pathways that contribute to postural control. These pathways may play an important role in trophic interactions between motoneurons and muscle fibers. Alterations of the gravitational field cause widespread effects in many behaviors and physiologic domains. For example, recent studies have shown that chronic exposure to altered gravitoinertial environments can alter otolith morphology and vestibulo-ocular reflex performance in developing zebrafish. The neuromuscular system responds to altered gravitational load with shifts in expression of myosin isoforms and physiologic 3 properties of muscle fibers. At the molecular level, exposure to microgravity (during space flight) can alter mRNA expression in a variety of adult mammalian tissues. A deeper understanding of interactions between gravity and mechanisms of gene expression would provide many benefits to the fields of developmental biology, space biology and space medicine.
The Workshop participants unanimously agreed that there is the need and opportunity to stimulate research utilizing specific, well-characterized trangenic (“knockout”/null expression, “knockin” and conditional expression) and mutant animal models to elucidate molecular bases for the normal development and function of sensorimotor mechanisms that detect and respond to gravity. The appropriate animal models include C. elegans, Drosophila, Xenopus laevis, zebrafish and the mouse, although other relevant models may emerge. Research in this area should address at least one of the following goals:
- Elucidate the molecular bases for functional development of sensory-motor mechanisms that sense and respond to gravitoinertial acceleration in a 1G environment
- Identify the molecular bases for functional maintenance and aging of normal sensory-motor function in a 1G environment
- Elucidate the influence of altered gravitoinertial environments on molecular bases for functional maintenance and aging of sensory-motor systems
- Develop conditional knockout/knockin and transgenic models with restricted spatial and temporal gene activation programs that address these issues
Example of studies that address these issues include, but are not limited to:
- Identification of the genetic bases for morphogenesis of the vestibular labyrinth
- Identification of the molecular bases for the development and maintenance of functional organization of sensory epithelia, including mechanisms regulating hair cell generation and normal establishment of the functional polarity of hair cells in the neuroepithelium
- Identification of the molecular bases for development of the afferent and efferent organization of vestibular neuroepithelia
- Identification of the molecular bases of and the influence of gravity on development of the cupulae and otoconia in the inner ear, including a clarification of the role of gravity in the formation and maintenance of otoconia
- Identification of the effects of maturation of vestibular and extra-vestibular graviceptive pathways on trophic interactions between motoneurons and muscle fibers
- Identification of the molecular bases for development and maturation of sensory-motor pathways in the central nervous system that sense and respond to gravity
- Characterization of the molecular mechanisms underlying altered performance of motor units and muscle fibers (e.g., atrophy and modified patterns of myosin expression) under altered gravitational conditions