During development of all multicellular organisms, the identity, movements, and ultimate function of cells must be coordinated with those of their neighbors. How do cells sense that they are located on the top, bottom, front, or back of an animal? How do cells differentiate into specialized tissues such as the gut and nervous system? How are these specialized tissues correctly wired and connected into a working animal, and what controls the timing of these processes? These are just a few of the key questions that developmental biologists would like to answer. In the past several decades, work on model organisms such as fruit flies, amphibians, fish, and mice has revealed a number of remarkable insights into these issues. To a large extent, it appears that many key developmental processes are guided by the release of highly conserved signaling molecules. These signals coordinate cellular development in time and space to achieve the wonderfully diverse set of specialized organs and marvelously adapted body plans that we see around us. We are interested in providing biochemical descriptions of how these signaling networks function, and how they have been adapted for different roles during development and evolution.
Early Axial Development
One of the earliest decisions that an embryo faces is assignment of coordinates along which the body plan will develop. The dorsal/ventral (D/V) axis runs from the back through the belly of the animal, and in flies, fish, and frogs there is remarkable conservation in the molecular mechanism that specifies this axis. In Drosophila, maternal factors guide the initial subdivision of the embryo along the D/V axis into the top and bottom halves. The top half, which will become the dorsal side of the embryo, comprises two tissues: the dorsal ectoderm, which gives rise to portions of the skin, and the amnioserosa, which guides the movements of gastrulation. Mutational analysis has identified at least five zygotic genes that guide the dorsal subdivision, and we have been investigating the biochemical mechanism behind this process.
The key gene products that pattern the dorsal half of the embryo are encoded by the decapentaplegic (dpp) and screw (scw) loci. Both Dpp and Scw are related to the bone morphogenetic proteins (BMPs) of vertebrates, which in turn are members of a large group of secreted factors known as the transforming growth factor-β (TGFβ) superfamily of signaling molecules. Members of this family control a wide range of developmental and physiological processes in higher eukaryotes.
One intriguing property of certain TGFβ-type proteins is their ability to act as morphogens—molecules that instruct cells to adopt specific fates in a concentration-dependent manner. The Drosophila Dpp protein has morphogen properties since, in the embryo, different levels of Dpp distinguish amnioserosa from dorsal ectoderm. How is the gradient of Dpp concentration generated in dorsal tissue, and how do the cells read the gradient to produce the appropriate response?
We have found that gradient formation is controlled at the extracellular level by interaction of Dpp and Scw with several other secreted factors, which include the products of the short gastrulation (sog), twisted gastrulation (tsg), and tolloid (tld) genes. The combined action of Sog, Tsg, and Tld localizes a heterodimer of Dpp/Scw in a tight distribution centered about the dorsal midline, leading to amnioserosa development, while the distributions of the Dpp/Dpp and Scw/Scw homodimers remain broad because they have low affinity for the Sog and Tsg proteins. The different ligand spatial profiles create a biphasic signal: amnioserosa precursors receive high levels of signal from the more potent Dpp/Scw heterodimer, while dorsal ectoderm cells are specified by the low levels of signal elicited by the Dpp/Dpp and Scw/Scw homodimers.
Although the basic features of the early D/V-patterning mechanism are now defined, many important properties of the system are poorly understood. For example, do heterodimers provide any selective advantage over homodimers in signaling systems? Do stochastic effects need to be considered in morphogen models? How do morphogen gradients adjust to properly pattern tissues of different sizes? How do cells choose a fate when the morphogen gradient changes during cell fate specification?
To answer these questions and others, we need to develop a quantitative description of the system. In collaboration with Hans Othmer (University of Minnesota), we have formulated a reaction-diffuse model of D/V patterning, and we are using computational methods to explore these issues. To function properly, despite differences in the genetic makeup and environmental conditions in which the organism develops, biological processes need to be fairly plastic. In such "robust" systems involved in the D/V-patterning process, heterodimers help protect the patterning system from changes in the concentration of the individual monomeric components. Thus, a 2-fold change in monomer input results in much less change in the effective concentration of the heterodimer. This may be one reason why heterodimers are employed by many biological processes.
TGFβs and Neuronal Plasticity
During the development of all animals, tissue growth must be coordinated for tissues to function properly as integrated units within the adult. This is especially important for wiring of the nervous system. To connect appropriately, axons must navigate over large distances, find a specific target cell, and then maintain appropriate synaptic efficacy during subsequent growth and development.
In the past few years, we have found that several different members of the TGFβ family play important roles in guiding different aspects of neuronal wiring and synapse remodeling. For example, during embryonic development, motor neurons send out processes in search of their appropriate muscle targets. Mutations in the activin-like ligand Dawdle disrupt the guidance of many motor axons en route to their muscle targets. The activity of this ligand is controlled by a metalloprotease, Tlr, that is related to the protease Tld, which controls early D/V patterning of the Drosophila embryo. In both cases, the metalloprotease destroys an inhibitory protein that enables the ligand to signal. In the case of Dawdle, this provides a permissive signal that controls axon guidance.
Once an axon finds its target, it must make and maintain a functional synapse. We have found that a second family of TGFβ factors, the BMPs, are involved in this process. In particular, Glass bottom boat (Gbb), a relative of the vertebrate BMPs 5/6/7 subfamily, signals retrogradely (from the muscle to the neuron) to regulate synaptic size and function at the Drosophila neuromuscular junction (NMJ). Mutations in gbb result in larvae with small synapses, reduced neurotransmission, and defects in the ultrastructure of synaptic active zones. These results reveal that target-derived BMP signals are required to coordinate muscle and synapse growth at the Drosophila NMJ.
Since synapse growth has been implicated as one means by which learning is accomplished in higher organisms, we are exploring whether BMP signaling regulates synaptic plasticity in the vertebrate brain. Our results using chordin-knockout mice (chordin is the vertebrate homolog of the Drosophila BMP inhibitor Sog) suggest that, as in the fly, BMP signals regulate aspects of synaptic plasticity. These studies should further our understanding of the signaling pathways that influence the process of learning and memory.
Regulation of Developmental Timing
Another important aspect of development is that regulatory processes must be properly timed so that cell growth and differentiation are appropriate for the particular life stage of the animal. In humans, for example, the transition from adolescence to adulthood is accompanied by rapid changes in growth and acquisition of sexual maturity. Likewise, in insects, developmental transitions also occur at regularly defined intervals. These transitions include molting, a process whereby the rigid exoskeleton is shed and resynthesized to accommodate increasing body size as a result of cell growth, and metamorphosis, a stunning transformation in which the immature larva changes into the reproductively mature adult. In both cases (human and insect) these changes are mediated to a large extent by small circulating steroid hormones that bind and activate transcription factors of the nuclear receptor superfamily. In many arthropods, 20-hydroxyecdysone (20E) is the primary active steroid hormone that coordinates developmental transitions. Although the genetic hierarchy that controls responses to 20E has received considerable attention, little is known about the genes involved in ecdysteroid biosynthesis and regulatory mechanisms that control hormone production.
To gain insight into developmental timing, we are interested in elucidating the mechanisms that regulate ecdysone biosynthesis and degradation. Since ecdysone is required to produce new cuticle at the various larval molts, we focused on a set of embryonic-lethal mutations that do not produce cuticle. We have cloned seven of these genes and found that six code for P450-type enzymes. In arthropods, ecdysteroids are synthesized from dietary cholesterol or phytosteroids via a series of hydroxylation steps catalyzed by P450-type enzymes. In collaboration with Lawrence Gilbert's laboratory (University of North Carolina), we have determined that mutations in these P450s all result in low embryonic titers of 20E or its immediate precursor, ecdysone. We have identified the exact biochemical step at which five of these enzymes act. More recently we been attempting to integrate the activity of these P450s is regulated. One key factor is the neuropeptide prothoracicotropic hormone (PTTH). We have identified a Drosophila PTTH homolog, and loss-of-function studies implicate this gene in regulating developmental timing, in part by controlling ecdysone biosynthetic P450 transcriptional levels. These studies should enable us to learn more about how key cellular and morphological changes are programmed to occur at appropriate times during development.