Gravity and Growth – Size Matters for Life on Earth
Size and Embryonic Growth
The shapes and forms that life might take on planets other than Earth has been assumed, both by scientists and novelists, to be ruled by gravity. If rocky planets of greater mass than Earth exist, then the life evolving under the resulting high gravity would be predicted to be short and squat, an ecosystem of diminutive animals of high musculature scuttling under stubby shrubs. In contrast, a planet small in size but still able hold on to an atmosphere would be imagined to be the home of tall, gracile creatures bounding under 300 foot forest canopies.
The animals that live at Earth’s 1g vary in size from single cell protozoa to 100 foot blue whales. Their size and mass are adaptations to the environmental niche in which they live. For instance, large delicate creatures, such as the invertebrate giant squid require the buoyancy of water to support their weight. Large land animals require high bone and muscle mass to support their weight. Under 1g gravity and 1000 millibars atmospheric pressure animals can be small enough to support their own weight in the air with wings to glide or fly. Each species is of a genetically predetermined size and weight to suit their environmental niche. Although this can vary somewhat depending on levels of nutrition, it is vital for the individual to be within a reasonable tolerance of its correct size. A 4 ft tall water buffalo would fare poorly amongst the rest of the herd whereas a 4 ft flying squirrel would be a falling squirrel!
A four-foot water buffalo meets a four-foot flying squirrel.
The variations that exist in size within each species are usually due to food availability as the animal develops. Variability can also occur during growth of the embryo, but it is important that this is limited because correct size is important at birth. If the animal is too small then its likelihood of surviving is much reduced; if too big then both pup and mother may die if the pup cannot pass easily through the birth canal. There are several reasons however why it may be useful to alter embryonic growth. If food is scarce or there exists an environmental stressor, such as a predator or toxic agent, then it would temporarily be advantageous to have small pups. If the number of embryos is large then, similarly, a small litter is better. Embryonic growth can also be moderated by genetic factors, in particular the insulin-like growth factors, a signaling system that provides a mechanism for parental control of embryonic size through imprinting (
http://www.informatics.jax.org/silver/5.5.shtml).
Changes in Growth under High Gravity Conditions
Initially, it may seem surprising that gravity is another factor that influences growth. Nevertheless many studies have shown that the growth rate of developing animals is slowed down when the pull of gravity is equivalent to double to that normally experienced. Such is the extent of this effect that rats bred under these conditions for three generations become progressively smaller. The growth inhibitory effects of gravity however are quite predictable given that size (or mass) is sensed via the judgment of weight and weight will rise with increasing gravity. It would be reasonable that, in compensation for the apparent increase in size, growth would slow down. Hoemeostasis is the maintenance of a constant internal condition, such as size. A homeostatic system is the mechanism by which this is maintained, and the systems that maintain the constant weight of the embryo are so far unknown. The effect could be at the organ level — for instance stretch receptors may be present in membranes that surround the embryo, which could detect an increasing pull as weight increased. Alternatively the effect could be at a cellular level. Although the way in which a single cell could detect an increase in its weight is not known, the suppressive effect of hypergravity on the growth of individual cells has been frequently reported and deformation of the internal skeleton (cytoskeleton) of the cell could provide a sensory system.
Experiments Showing the Effects of Hypergravity on Growth and Gene Expression
Our project investigates the genes involved in detecting and affecting these responses to increased gravity. A question at this point might be how gravity can be manipulated without travel to a larger planet or by breaking the laws of physics and using a gravity generator. The answer is to create conditions that mimic gravity using centrifugal force (
http://lifesci.arc.nasa.gov/cgbr/home.html). We have used the 24-foot centrifuge situated at NASA-Ames in California (
http://lifesci.arc.nasa.gov/cgbr/24_ft_cent.html) (see below) that allows caged, pregnant animals to be spun at an equivalent force of 2.0g for up to weeks at a time. This simulates high gravity (hypergravity) conditions.
The 24-foot centrifuge situated at NASA-Ames, California
Mice have a gestation period of 19 days and development of the nervous system starts from embryonic day 7. We centrifuged pregnant mice from embryonic day 4 up to day 14. We are using a series of techniques including gene array and Northern blotting to analyze changes in gene expression in the whole embryo and in the brain. Measuring gene expression tells us which genes are actively encoding proteins that can be used to change the cell’s behavior, e.g. from a growing to a non-growing state. We intend to identify genes that are altered in level of expression as part of the response to increased gravity anticipating that these will be either part of the initial sensing of hypergravity or components of the machinery to reduce growth rate.
We started our analysis at an early point of brain development, embryonic day 10 and compared embryos after 6 days of centrifugation to non-centrifuged controls. To provide an initial screen to identify which genes change as a result of 2g centrifugation we used the gene array technique (
http://www.gene-chips.com/ ). Gene arrays can identify changes in expression of large numbers of genes in a single experiment. The level of gene activity is determined by measuring the amount of mRNA that is transcribed from a particular gene — mRNA is the intermediary between the gene and the protein that it encodes messenger RNA (mRNA)
We have screened for two types of genes — those that change in response to stress and those that are part of the cell cycle. Genes that respond to stress may be part of the means to detect hypergravity; genes of the cell cycle are those that allow the cell to divide and hence are necessary for growth of the organism.
The first striking result of these experiments is that very few genes change in their expression. This indicates that the effect of gravity is not a generalized stress such as occurs when food intake is limited leading to an overall reduction in protein synthesis. We find however a decrease in three genes involved in the cell cycle: CDK 5 decreasing by 165%, p15INK4b falling by 192% and Gadd45 dropping by a tremendous 1450%.
Gene array showing the expression of 62 genes
in embryonic day 10 embryos. Of these only three are
reduced in gene activity comparing centrifuged (right)
versus control (left); CDK5, p15INK4b and Gadd45a.
These results imply that there is significant change in cell growth in the centrifuged animals. This is reflected in a decrease in size in these embryos – at embryonic day 10 the control animals were 3.9 mm whereas the centrifuged embryos had only reached a length of 2.7 mm. It is clear then that centrifugation depresses the embryos rate of growth and reduces the activity of certain genes that influence the cell cycle. Could this suppression of growth extend to later periods of development and affect the birth of mature cells, such as neurons? Such a result had not been previously described but, given the general effects of centrifugation on growth, this was a possibility. In this case we determined the level of gene activation (the amount of mRNA transcribed from a particular gene) by Northern blotting
This type of experiment separates the mRNA by size driving the molecules with an electric current through the small pores of a gel, the small mRNAs moving fastest as a band, the large mRNAs moving slower. The mRNAs of interest are identified by labeled probes of complimentary sequence to the mRNA. The probes bind very tightly and specifically and the labeling means that they can be visualized. The figure below shows a Northern blot for NeuroD, a gene that is activated as neurons are born. The embryos were centrifuged for 8 days and gene expression in the brain measured at embryonic day 12 a time at which many neurons are born. In the centrifuged sample when compared to several different types of non-centrifuged controls, there is a large decrease in the activity of this gene. This implies that there is a decrease in the birth of new neurons.
Northern blot showing a decline in gene activity of the NeuroD gene in centrifuged animals (Exp, top panel) compared to control groups Cs,Cu and Cf (top panel). The lower panel shows that a control gene does not vary between the samples.
Conclusions So Far…
The purpose of this project is to understand how growth is regulated in the developing embryo under conditions of 2g hypergravity, conditions which deceive the embryo into sensing that it is twice its mass. The initial results indicate that several genes involved in regulating cell division are reduced during early development (embryonic day 10). At later developmental stages, a decline in the NeuroD gene necessary for neuronal birth suggests that there may also be a decline in the number of neurons generated in the brain. This approach will help to comprehend the normal mechanisms of growth homeostasis and also to understand how they may go wrong. Many birth abnormalities, including those that include mental retardation, involve changes in growth rate and size – whether of the embryo as a whole or limited to the central nervous system. These diseases include Down syndrome, Fetal Alcohol Syndrome, intrauterine growth restriction and deficiencies in iron, iodine or vitamin A. We expect that there will be some common pathways shared between these syndromes. The elucidation of genes involved in the regulation of embryonic size and mass will help to identify and characterize these pathways.
