A goal of neural development studies is to understand how patterns of gene expression are translated into the cellular diversity and patterns of cellular connections that characterize complex nervous systems. We are focusing on neuronal stem cells in the central nervous system (CNS) of Drosophila melanogaster to make the linkage between gene expression and connectivity and behavior. The CNS of the fruit fly is produced from a fixed number of neuronal stem cells termed neuroblasts, which go through repeated asymmetric divisions. The smaller product of each division is the ganglion mother cell (GMC), which then divides once to produce two daughters, "A" and "B." Most neuroblasts produce an initial, small set of diverse neurons that make the CNS of the larva, and then a much larger set of neurons to be used in the adult. The latter cells, termed secondary neurons, constitute discrete classes of neurons having similar properties.
We are currently focusing on the secondary neurons in the segmental CNS of Drosophila. Although there are about 200 neuroblasts that produce approximately 20,000 secondary neurons for this portion of the CNS below the brain, these represent a reiterated set of only 25 different types of stem cells. Each of these 25 stem cells is programmed to generate two classes of secondary neurons, based on the A and B daughters arising from the division of the GMC. The secondary neurons having the same daughter cell identity within a lineage are termed the hemilineage, and neurons within a hemilineage share common patterns of molecular expression, pathfinding choices, and initial targets.
Prior to metamorphosis, the neurons produced by each stem cell form a compact cluster and are arrested at the same point in development. We have concentrated on assessing the patterns of molecular expression at this point and relating these to early decisions of identity, pathfinding, and neuropil partitioning. We have initially focused on the thoracic region of the CNS to generate lineage-based approaches but intend to utilize this approach in the brain also.
Generation of Neuronal Phenotypes
We are especially interested in transcription factors that provide molecular signatures for specific neuroblasts, their GMCs, or their daughters. We have identified core GMC genes that are expressed in all of the GMCs in a lineage, core lineage genes that are expressed in all of the daughter neurons, and core hemilineage genes that are expressed in only the A or the B daughters. We use both loss- and gain-of-function approaches to determine the role of these genes in controlling neuronal identity and development. We find that core GMC genes are typically involved in establishing the identity of one or both of the sibs, and mutation of these genes results in a major shift in sib identity. For the core hemilineage genes, by contrast, loss of function does not cause an obvious change in identity, but rather selected aspects of the neuron's phenotype are altered; for example, the mutated neurons navigate to novel initial targets but maintain the other properties characteristic of that type of neuron. For a few representative core GMC genes and a few core hemilineage genes, we are exploring the downstream pathways responsible for the identity or phenotypic changes.
We are continuing to map the expression patterns of transcription factors onto the segmental lineages and are testing the roles of any genes that show core GMC or core hemilineage expression. We are interested in determining if there are overarching rules relating GMC or lineage/hemilineage expression to pathfinding or wiring decisions, or if there are 25 ad hoc patterns, each associated with 1 of the 25 neuroblasts and their progeny.
We have also been identifying transcription factors that are expressed in all lineages but during restricted temporal windows. We already have two such genes that serve as molecular "time stamps." They are expressed in all lineages, but only in those cells that are born during specific windows of larval life. Once induced, they remain stably expressed into the adult. Therefore, they define discrete molecular subclasses within each hemilineage. We are interested in identifying more of these genes and whether they establish phenotypic, as well as molecular, subclasses within the hemilineages. In collaboration with Lynn Riddiford (HHMI, Janelia Farm Research Campus), we also intend to establish the role of extrinsic (hormonal?) signals in regulating their expression.
Development of Lineage-Based Tools for Studying CNS Function
The key to using a hemilineage-based approach to study CNS development and function is to have a set of driver lines that can target gene expression to single, defined hemilineages. With this aim in mind, we are collaborating with Gerald Rubin (HHMI, JFRC) in screening his collection of GAL4 lines to find lines that show expression restricted to specific lineages or hemilineages of secondary neurons. We are especially interested in driver lines that begin to be expressed when a neuron is born and remain on into the adult stage. A goal is to have a library of such lines that cover all of the hemilineages so that we can direct gene expression to alter the development or function of defined classes of central neurons.
We are also interested in the various binary expression systems that are being developed. Having one component that is hemilineage restricted and a second that is based on time of cell birth would give us the ability to be exquisitely specific in our genetic manipulations of development or behavior.
Construction of Adult Neuropils
A long-term goal is to link the early molecular expression in the secondary neurons to the circuits that they construct. We are initially concentrating on the thoracic leg neuropil because it is constructed de novo within the larval CNS, entirely from the secondary neurons. Seventeen hemilineages, two of which make just motor neurons, are exclusively devoted to making each neuropil, and these provide the classes of neurons that form the computational circuitry for the leg. This neuropil begins as a partitioned neuropil, with the endings from each hemilineage occupying exclusive domains within the forming structure. Boundaries between hemilineages are maintained until the start of metamorphosis, when steroid signals cause exuberant sprouting of the immature neurons and the partitions disappear. An important question is how the neighbors at partition boundaries relate to the final synaptic partners. It would be exciting if the initial contacts in the early developing neuropil prefigure the major synaptic partners seen in the mature CNS.
We think that the leg neuropil provides an excellent opportunity to understand the rules that govern the construction of a complex neuropil that deals with information flow from sensory processing to motor patterning. By using the GAL4 lines described above, we will be able to alter the numbers and/or properties of each class of neuron in the leg control circuit selectively and then use behavioral and imaging approaches to assess the effects on leg movement. In collaboration with Julie Simpson (HHMI, JFRC), we will then move this approach into the adult brain.
Macro- and Microevolution of the CNS
By focusing on the hemilineages, we can assess individual variation in the neuronal composition in the CNS and how this variation may relate to behavior. Since we think that the secondary neurons in a given class have broadly overlapping functions, alterations in the numbers of these neurons would likely increase or decrease the temporal and spatial computation within the network to which they belong. As we complete the collection of molecular markers for each hemilineage, we can ask, how constant is the number of neurons generated by a given stem cell? Do numbers change with fly populations or in response to environmental factors during the larval growth? How does this variation relate to function? For example, if flies have been selected for faster running, do we see increased numbers of neurons in the leg-related hemilineages, but no change in numbers in the flight-related hemilineages?
We are also interested in broader issues relating to the evolution and diversification of the CNS. After we characterize the neurons made in each of the hemilineages in Drosophila, we can then ask how stable these phenotypic classes have been through evolution. Do the neurons that are made by the homologous stem cells in other insects, such as moths, beetles, and bees, have the same pattern of initial projections and the same transmitter phenotypes as seen in the fly? For the lineages that differ significantly, we would be interested in how this difference is reflected in molecular expression, especially of core GMC, lineage, and hemilineage genes. If there are molecular differences (e.g., the moth lineage has a transcription factor that is not expressed in the homologous fly lineage, or vice versa), can we then go to Drosophila to make the appropriate molecular changes and produce moth-type neurons in the fly CNS?
We are also intrigued by our finding that the fly makes a number of classes of secondary neurons that all die. We suspect that these neuronal classes are used in basal insects but are lost in the highly derived CNS of Drosophila. Can we find these neurons in other insects, and what are their phenotypes and function? Although they are not normally present in flies, we can study their function in Drosophila by genetically blocking their death. Do they then become incorporated into the adult circuitry, and, if so, how does their presence impact the development and functioning of other lineages and of the CNS as a whole?