This work will study the mechanisms that regulate the proportional sizes of body parts. The relationship between the relative sizes of body parts and overall body size is called allometry. Within a taxonomic group of animals, morphological evolution occurs primarily by changes in allometry. Hence understanding the processes by which body parts achieve their absolute and relative sizes is critical for a deep understanding of the development and evolution of animal form. Most experimental work in this field has focused on "bottom up" studies that examine the effects of the genetic manipulation on the final size. What is missing is an understanding of the processes by which body parts get to their exact final size and shape. The "top down" approach proposed here will reconstruct the pathways by which body parts achieved their final size by studying how local cell proliferation, cell enlargement, and cell death act to determine final size and shape. The proposed work will focus on the development of the wings of insects where cellular studies are particularly tractable. The proposed work will discover whether genetically- and nutritionally-caused differences in absolute and relative wing sizes and shapes are due to differences in cell size or cell number, in localized rate of cell divisions and/or the timing of cell divisions, and patterned cell death. The significance of this work is that it will begin to identify, step by step, the underlying processes by which genetic changes lead to changes in size and shape.
Broader Impacts The approach to understanding size and shape proposed here is generally applicable to other body parts and other species and is an essential complement to molecular-genetic approaches. The education of undergraduate students has always been an integral part of Dr. Nijhout's research program. The research of undergraduate students has resulted in 7 publications in the peer-reviewed literature in the past 5 years. Dr. Nijhout gives regular public lay person's lectures about his NSF-funded research at various venues in the Durham area.
Aims of the research. Size and shape are the defining characters of animal species, yet understanding how the species-specific body size and the relative sizes of body parts are regulated continues to be one of the great unsolved puzzles in Biology. The aim of this research program was to investigate the developmental mechanisms in insects by which body size and the relative sizes of body parts are controlled. Our work has shown that body size is determined by the physiological mechanism that controls exactly when growth will stop. The elucidation of this mechanism was one of the great accomplishments of this grant period. Another significant accomplishment was the elucidation of the mechanism by which wings are controlled to grow to the correct proportion with body size, and how the shape of the wing is controlled at the cellular level. How body size is sensed. The problem of size regulation is the two-fold problem of (1) how to asses body size and (2) how to stop growing when the right, species-characteristic, body size is reached. We found that the size of the tracheal system (the respiratory system of insects: a system of air-filled tubes that take air directly to every cell in the body) is fixed at the beginning of the last larval instar and does not grow, even though the larva continues to grow and increase in size ten-fold. As larva grows there is a point at which the tracheal system becomes unable to keep up with the increasing oxygen demand of the growing body. The point at which this occurs corresponds to the critical weight at which the decision to stop growing is made. Accordingly, we also found that when grown in an atmosphere with reduced oxygen levels insects stop growing at a smaller size. We then showed that oxygen restriction acts as a signal to initiate the secretion of the hormones that terminate the growth phase and initiate metamorphosis. This is the first demonstration of a mechanism for sensing body size in development. Wing-body scaling. Body size is also controlled by nutrition. Animals that are underfed grow to a much smaller size than animals that are fed normally. We showed that most of the growth of the wings occurs after the body has stopped growing, and studied the mechanism by which the growth and size of the wings is adjusted to correctly match the size of the body in animals that varied in size due to variation in nutrition. We found that the control of wing size occurs by adjusting the number of cell divisions in the wings during a very brief period early in their development, and that this pattern of cell divisions is controlled by ecdysone, a steroid hormone. The secretion of ecdysone is adjusted to body size so that in smaller animals ecdysone stimulates fewer cell divisions and less growth than in larger animals. This is the first and only case in which we understand how the growth and size of body parts is accommodated to be proportional to the size of the body. Development of wing shape. Wing-body scaling is not simply a matter of having a wing of the correct "size." The wing also has to be the right species-specific "shape." To study the developmental mechanism that produce species-specific wing shapes we undertook a comparative study of two species with very different wing shapes and sizes. We analyzed the growth of wings at the cellular level and found that there are species-specific spatial patterns of density of cell divisions that change throughout the development of the wing and that precisely regulate the changing patterns of growth through which a wing gradually achieves its species-specific shape. Conclusions. The work supported by this grant has given us several novel insights into the developmental and physiological mechanisms that control size and shape. Using a multi-pronged analysis at the cellular, endocrine and physiological levels we discovered the mechanism by which body size is sensed, the mechanism that stops growth when the right size is reached, the mechanism by which wings develop in correct proportion to the body, and the mechanism that produces the species-specific shape of the wing
