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Postnatal development under conditions of simulated weightlessness and space flight

The adaptability of the developing nervous system to environmental influences and the mechanisms underlying this plasticity has recently become a subject of interest in space neuroscience. Ground studies on neonatal rats using the tail suspension model of weightlessness have shown that the force of gravity clearly influences the events underlying the postnatal development of motor function. These effects depend on the age of the animal, duration of the perturbation and the motor function studied. A nine-day flight study has shown that a dam and neonates can develop under conditions of space flight. The motor function of the flight animals after landing was consistent with that seen in the tail suspension studies, being marked by limb joint extension. However, there were expected differences due to: (1) the unloading of the vestibular system in flight, which did not occur in the ground-based experiments; (2) differences between flight and suspension durations; and (3) the inability to evaluate motor function during the flight. The next step is to conduct experiments in space with the flexibility and rigor that is now limited to ground studies: an opportunity offered by the International Space Station. Copyright 1998 Published by Elsevier Science B.V.

Non-NASA Center

Models and molecular approaches to assessing the effects of the microgravity environment on vertebrate development

The extent to which gravity, and especially the lack thereof, can affect normal development in higher organisms is poorly understood. Underlying this question is the assumption that normal development depends on the embryo's ability to maintain a programmed temporal and spatial coordination of morphogenetic events. There are several reports documenting the apparently normal development of several vertebrate species, including mammals, under conditions of exposure to space flight during various periods of the development process. Evidence to the contrary also exists and it is therefore likely that some alterations in morphology do occur in a microgravity environment. Although subsequent development may appear overtly normal, more subtle abnormalities result. In all studies, the evaluation is restricted by the few numbers of specimens that can be examined and the relatively insensitive techniques for assessing potentially subtle effects. In the present discussion, we summarize some observations of mammalian development made in microgravity and consider which stages might be expected to be differentially sensitive to altered gravity conditions. While we emphasize mammalian development, we discuss the suitability of another model system for examining such effects in a cross-species context. Furthermore, we consider recent developments in our understanding of the molecular genetic program regulating embryogenesis that could serve as markers for assessing perturbations of development.

Cosmos 1129 Project

Eye development and the appearance and maintenance of corneal transparency

Embryonic development of the eye, including the cornea, depends on the appearance and steady maintenance of intraocular pressure. The eye is a gravity-sensitive organ, as evidence by changes in pupil diameter during parabolic flight. The cornea is largely a paracrystal of extracellular matrix. The extent to which it will polymerize normally in microgravity has yet to be determined.

NASA Discipline Developmental Biology

Collagen in organ development

It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

NASA Discipline Cell Biology

Extracellular matrix and growth factors in branching morphogenesis

The unifying hypothesis of the NSCORT in gravitational biology postulates that the ECM and growth factors are key interrelated components of a macromolecular regulatory system. The ECM is known to be important in growth and branching morphogenesis of embryonic organs. Growth factors have been detected in the developing embryo, and often the pattern of localization is associated with areas undergoing epithelial-mesenchymal interactions. Causal relationships between these components may be of fundamental importance in control of branching morphogenesis.

Non-NASA Center

Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall

Neurons destined for each region of the neocortex are known to arise approximately in an "inside-to-outside" sequence from a pseudostratified ventricular epithelium (PVE). This sequence is initiated rostrolaterally and propagates caudomedially. Moreover, independently of location in the PVE, the neuronogenetic sequence in mouse is divisible into 11 cell cycles that occur over a 6 d period. Here we use a novel "birth hour" method that identifies small cohorts of neurons born during a single 2 hr period, i.e., 10-20% of a single cell cycle, which corresponds to approximately 1.5% of the 6 d neuronogenetic period. This method shows that neurons arising with the same cycle of the 11 cycle sequence in mouse have common laminar fates even if they arise from widely separated positions on the PVE (neurons of fields 1 and 40) and therefore arise at different embryonic times. Even at this high level of temporal resolution, simultaneously arising cells occupy more than one cortical layer, and there is substantial overlap in the distributions of cells arising with successive cycles. We demonstrate additionally that the laminar representation of cells arising with a given cycle is little if at all modified over the early postnatal interval of histogenetic cell death. We infer from these findings that cell cycle is a neuronogenetic counting mechanism and that this counting mechanism is integral to subsequent processes that determine cortical laminar fate.

NASA Discipline Cell Biology

Mineralization and growth of cultured embryonic skeletal tissue in microgravity

Microgravity provides a unique environment in which to study normal and pathological phenomenon. Very few studies have been done to examine the effects of microgravity on developing skeletal tissue such as growth plate formation and maintenance, elongation of bone primordia, or the mineralization of growth plate cartilage. Embryonic mouse premetatarsal triads were cultured on three space shuttle flights to study cartilage growth, differentiation, and mineralization, in a microgravity environment. The premetatarsal triads that were cultured in microgravity all formed cartilage rods and grew in length. However, the premetatarsal cartilage rods cultured in microgravity grew less in length than the ground control cartilage rods. Terminal chondrocyte differentiation also occurred during culture in microgravity, as well as in the ground controls, and the matrix around the hypertrophied chondrocytes was capable of mineralizing in both groups. The same percentage of premetatarsals mineralized in the microgravity cultures as mineralized in the ground control cultures. In addition, the sizes of the mineralized areas between the two groups were very similar. However, the amount of 45Ca incorporated into the mineralized areas was significantly lower in the microgravity cultures, suggesting that the composition or density of the mineralized regions was compromised in microgravity. There was no significant difference in the amount of 45Ca liberated from prelabeled explants in microgravity or in the ground controls.

NASA Discipline Developmental Biology

Calcium-binding proteins and development

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

Review

A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium

Neuronogenesis in the neocortical pseudostratified ventricular epithelium (PVE) is initiated rostrolaterally and progresses caudo-medially as development progresses. Here we have measured the cytokinetic parameters and the fractional neuronal output parameter, Q, of laterally located early-maturing regions over the principal embryonic days (E12-E15) of neocortical neuronogenesis in the mouse. These measures are compared with ones previously made of a medial, late-maturing portion of the PVE. Laterally, as medially, the duration of the neuronogenetic interval is 6 days and comprises 11 integer cell cycles. Also, in both lateral and medial areas the length of G1 phase (TG1) increases nearly 4-fold and is the only cell cycle parameter to change. Q progresses essentially identically laterally and medially with respect to the succession of integer cell cycles. Most importantly, from E12 to E13 there is a steeply declining lateral to medial gradient in TG1. The gradient is due both to the lateral to medial graded stage of neuronogenesis and to the stepwise increase in TG1 with each integer cycle during the neuronogenetic interval. To our knowledge this gradient in TG1 of the cerebral PVE is the first cell biological gradient to be demonstrated experimentally in such an extensive proliferative epithelial sheet. We suggest that this gradient in TG1 is the cellular mechanism for positionally encoding a protomap of the neocortex within the PVE.

Non-NASA Center

A mechanism of adaptation to hypergravity in the statocyst of Aplysia californica

The gravity-sensing organ of Aplysia californica consists of bilaterally paired statocysts containing statoconia, which are granules composed of calcium carbonate crystals in an organic matrix. In early embryonic development, Aplysia contain a single granule called a statolith, and as the animal matures, statoconia production takes place. The objective of this study was to determine the effect of hypergravity on statoconia production and homeostasis and explore a possible physiologic mechanism for regulating this process. Embryonic Aplysia were exposed to normogravity or 3 x g or 5.7 x g and each day samples were analyzed for changes in statocyst, statolith, and body dimensions until they hatched. In addition, early metamorphosed Aplysia (developmental stages 7-10) were exposed to hypergravity (2 x g) for 3 weeks, and statoconia number and statocyst and statoconia volumes were determined. We also determined the effects of hypergravity on statoconia production and homeostasis in statocysts isolated from developmental stage 10 Aplysia. Since prior studies demonstrated that urease was important in the regulation of statocyst pH and statoconia formation, we also evaluated the effect of hypergravity on urease activity. The results show that hypergravity decreased statolith and body diameter in embryonic Aplysia in a magnitude-dependent fashion. In early metamorphosed Aplysia, hypergravity decreased statoconia number and volume. Similarly, there was an inhibition of statoconia production and a decrease in statoconia volume in isolated statocysts exposed to hypergravity in culture. Urease activity in statocysts decreased after exposure to hypergravity and was correlated with the decrease in statoconia production observed. In short, there was a decrease in statoconia production with exposure to hypergravity both in vivo and in vitro and a decrease in urease activity. It is concluded that exposure to hypergravity downregulates urease activity, resulting in a significant decrease in the formation of statoconia.

NASA Program Space Biology

Development of the otolith organs and semicircular canals in the Japanese red-bellied newt, Cynops pyrrhogaster

The sequence in which the otoliths and semicircular canals and their associated sensory epithelia appear and develop in the newt are described. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos from stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. A single otolith is first seen in stage 33 embryos (approximately 9 days old); this splits into separate utricular and saccular otoliths at stage 40 (13 days). The lateral semicircular canal is the first to appear, at stage 41 (14 days). The anterior and posterior canals appear approximately one week later and the vestibular apparatus is essentially fully formed at stage 58 (approximately 5 weeks). The data reported here will serve as ground-based controls for fertilized newt eggs flown on the International Microgravity Laboratory-2 Space Shuttle flight, to investigate the influence of microgravity on the development of the gravity-sensing organs.

NASA Discipline Neuroscience

The effect of spaceflight on retino-hypothalamic tract development

Researchers examined the effect of late prenatal exposure to microgravity on the development of the retina, retinohypothalamic tract, geniculo-hypothalamic tract, and suprachiasmatic nucleus. Results indicate an effect on c-fos activity in the intergeniculate leaflet between gestational day 20 and postnatal day 8, suggesting a delay in development of the circadian timing system.

Non-NASA Center

Histological preparation of developing vestibular otoconia for scanning electron microscopy

The unique nature of vestibular otoconia as calcium carbonate biominerals makes them particularly susceptible to chemical deformation during histological processing. We fixed and stored otoconia from all three otolith endorgans of embryonic, hatchling and adult Japanese quail in glutaraldehyde containing either phosphate or non-phosphate buffers for varying lengths of time and processed them for scanning electron microscopy. Otoconia from all age groups and otolith endorgans processed in 0.1 M phosphate buffer (pH 7.4) showed abnormal surface morphology when compared to acetone fixed controls. Otoconia processed in 0.1 M sodium cacodylate or HEPES buffered artificial endolymph (pH 7.4) showed normal morphology that was similar to controls. The degree of otoconial deformation was directly related to the time exposed to phosphate buffer. Short duration exposure produced particulate deformations while longer exposures resulted in fused otoconia that formed solid sheets. Otoconial surface deformation and fusing was independent of the glutaraldehyde component of the histological processing. These findings should help vestibular researchers to develop appropriate histological processing protocols in future studies of otoconia.

Non-NASA Center