Yuh-Nung Jan, Ph.D.
We are interested in the basic mechanisms of neural development. Our strategy is to use the relatively simple Drosophila peripheral nervous system (PNS) to discover the genetic program that controls its development. In doing so, we hope to uncover evolutionarily conserved core programs that control different steps of neural development in animals. We started with the earliest steps in neural development (neurogenesis and neuronal cell fate specification) and gradually worked our way toward later steps (neuronal morphogenesis and the assembly of a functional neuronal circuit). Some highlights of our earlier efforts include the finding of atonal and numb: atonal is the founding member of a family of proneural genes that initiate the development of two major types of sensory neurons used in vision and hearing; numb functions as a cell fate determinant during asymmetric cell division. Numb provided a starting point for the study of asymmetric cell division in Drosophila and vertebrates, which led to considerable insight into the molecular basis of asymmetric cell division. In recent years, the major focus of our lab has shifted to the study of dendrite development and neuronal circuit assembly.
Genetic Control of Dendrite Development in Drosophila
Dendrite arborization patterns are critical determinants of neural circuit formation and influence the type of synaptic or sensory inputs a neuron is able to receive. Moreover, dendrite defects are associated with a variety of known human mental disorders, such as autism. Relatively little is known about the molecular mechanisms that control dendrite development. To use Drosophila genetics to identify core programs that control dendrite development, we developed a simple assay system. We use the fly transgenic technique to express green fluorescent protein (GFP) in the dendritic arborization (da) neurons, a group of sensory neurons with a stereotyped dendritic branching pattern. This allows us to visualize the development of the dendrites of da neurons in the living fly embryos and to use them as an assay system for a genetic dissection of dendrite development. We have made some recent progress:
1. Regulation of dendritic field size and complexity by transcription factors. Transcription factors are important regulators of the size and complexity of dendritic fields, and the logic of their usage is beginning to emerge. In some cases, the "dendritic fate" of a particular neuron might be specified by a single transcription factor. For example, in the Drosophila PNS, the zinc finger–containing protein Hamlet functions as a binary switch between the elaborate multiple-dendrite morphology of the da neuron and the single, unbranched dendrite morphology of the external sensory (es) neuron. In most cases, however, the dendritic fate is determined by the combined action of multiple transcription factors. Drosophila da neurons fall into four distinct morphological classes (I–IV). The selector gene cut is expressed at different levels in the da neurons. Neurons with small and simple dendritic arbors either do not express Cut (class I neurons) or express low levels of Cut (class II). Neurons with more complex dendritic branching patterns and larger dendritic fields (classes III and IV) express higher levels of Cut. Cut levels are a critical determinant of da neuron class-specific dendritic morphologies.
In contrast to Cut, Spineless (Ss), the homolog of the mammalian dioxin receptor, is expressed at similar levels in all da neurons. In ss mutants, different classes of da neurons elaborate dendrites with similar branch numbers and complexities, suggesting that da neurons might reside in a common "ground" state in the absence of ss function. Studies of the epistatic relationship between Cut and Ss indicate that these transcription factors are likely acting in independent pathways to regulate morphogenesis of da neuron dendrites. These findings, together with an analysis of an RNAi (RNA interference) screen that revealed more than 70 transcription factors regulate dendritic arbor development of class I neurons in fly, suggest that complicated networks of transcriptional regulators likely regulate type-specific dendrite arborization patterns.
2. The molecular mechanism for dendritic self-avoidance and tiling. Dendrite-dendrite repulsion can have a profound influence on the size and shape of the dendritic field, as well as the spatial relationship between different dendritic fields. The dendrites of each da neuron show self-avoidance and tend to spread out without crossing over. Class III and class IV da neurons show tiling; i.e., there is little overlap between the dendritic fields of adjacent neurons of the same class because their dendrites show homotypic repulsion. Recently we found that Dscam (Down syndrome cell adhesion molecule), originally identified as axon guidance receptors by Lawrence Zipursky (HHMI, University of California, Los Angeles) and his colleagues, is needed for self-avoidance and contribute to the spreading of dendrites. Without Dscam, the dendrites of each da neuron bundle together or cross over. For the dendritic fields of different neurons to coexist in the same space, they need to express different Dscam isoforms. In contrast, tiling requires some cell surface recognition molecules other than Dscam to mediate the homotypic repulsion. Although the signals that mediate tiling behavior remain elusive, the evolutionarily conserved protein kinase Tricornered (Trc) and the putative adapter protein Furry (Fry) have been identified as important components of the intracellular signaling cascade involved in tiling. In trc or fry mutants, dendrites no longer show their characteristic turning or retracting response when they encounter same-type dendrites.
3. Dendrite-specific developmental regulators. From our mutant screen, we identified a group of dar (dendritic arborization reduction) genes. Mutations of any of the dar genes lead to defective dendritic arbors but normal axonal projections. Thus, studies of dar genes should reveal how axons and dendrites are made differently. We estimate that there may be a total of about 20 dar genes in Drosophila. All five of the dar genes that we have cloned so far have mammalian homologs. Remarkably, three encode components of the secretory pathway. These results reveal the preferential role of endoplasmic reticulum–Golgi trafficking and Golgi outposts in dendrite arborization.
4. The maintenance of dendritic fields. Our genetic screen revealed specific mechanisms that ensure maintenance of dendritic arbors. We found that the tumor-suppressor Warts (Wts), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila (the other being Trc), and the Polycomb group of genes are required for the maintenance of class IV da dendrites. Loss-of-function mutants of any of those genes cause a progressive defect in the maintenance of dendritic tiling, resulting in large gaps in the receptive field. How are establishment and maintenance of dendritic fields coordinated? In Drosophila class IV neurons, the Ste-20–related tumor-suppressor kinase Hippo (Hpo) can directly phosphorylate and regulate both Trc, which functions in the establishment of dendritic tiling, and Wts, which functions in the maintenance of dendritic tiling. How Hpo regulates the transition from establishment to maintenance of dendritic fields remains to be determined.
5. The remodeling of dendritic fields. Drosophila class IV da neurons undergo dramatic remodeling during metamorphosis. Early in the pupal stage, those neurons prune all their dendrites. Later each neuron grows a completely new dendrite for adult function. While the dendrites are being remodeled, the axons stay largely intact. We have begun to identify the molecular mechanisms that control this large-scale, dendrite-specific remodeling.
Extension of Our Drosophila Work to Dendrite Development of Mammalian Central Neurons
The great majority of the genes found to affect Drosophila dendrite development have a mammalian homolog(s). In several cases, we have shown that those homologs (for example, Dasm1, Dar3/Sar1) have similar function in regulating dendrite development in the mammalian central nervous system. We will continue to extend our findings about dendrite development in Drosophila to the mammalian nervous system by studying cultured hippocampal neurons or knockout mice.
Grants from the National Institutes of Health provided partial support for our research.