Wiring brains requires routing axons and dendrites to appropriate regions, such as layers and columns, to form correct synaptic connections during development. Many neuropsychiatric disorders, such as Down syndrome, Fragile X syndrome and Rett syndrome, have development origins and exhibit abnormal dendritic morphological defects, such as changes in branching numbers and patterns. Dendritic defects could cause neuronal connectivity defects, which likely underline neurological and cognitive deficits. It remains unclear, however, how genetic disorders lead to dendritic patterning defects during development, leading to erroneous connections and functional deficits in adults. In this project, we use Drosophila optic lobe neurons as a model to study dendrite development and neural circuit assembly in the central nervous system. Like vertebrate cortex and retina, the Drosophila optic lobe is organized in columns and layers, suggesting that the fly visual neurons and vertebrate cortex neurons face similar challenges in routing their dendrites to specific layers and columns during development. In addition, the Drosophila visual system has several unique advantages: (i) the medulla neurons extend dendritic arbors in a lattice-like structure, facilitating morphometric analysis; (ii) the synaptic partnership is known; (iii) genetic tools for labeling specific classes of medulla neurons and determining their connectivity are available; (iv) sensitive behavioral assays are available for quantifying functional deficits. To analyze the dendritic patterns of the medulla neurons, we developed a number of techniques: (i) a dual-imaging technique for high-resolution imaging; (ii) a registration technique to standardize and to compare dendritic patterns; (iii) two modified GRASP methods for visualize synaptic connections at the light-microscopic level; (iv) a two-tag dual labeling method for examining synapses at the electron microscopic level. Using these techniques, we first analyzed the dendritic morphologies of four types of medulla neurons, Tm1, Tm2, Tm9 and Tm20. We identified four dendritic attributes: (i) layer-specific dendritic initiation, (ii) planar projection direction, (iii) layer-specific dendritic termination, (iv) type-specific size of dendritic receptive field. (i) For Tm1/2/9/20, most (>75%) dendritic branches are originated from one or two primary branching nodes on the axons in the medulla M2/3 layers, while the other Tm neurons, such as Tm5a/b, initiate their dendrites in the M5/6 layers. (ii) Tm1/2/9 project their dendrites anteriorly to innervate their cognate columns while Tm20 dendrites project posteriorly. (iii) Tm1/2/9/20 project dendrites to terminate in specific layers in a type-specific fashion that matches their presynaptic partners. (iv) The dendrites of four types of Tm neurons are largely confined in single medulla columns while the amacrine neurons Dm8 extend a large dendritic tree to cover about 14 medulla columns. By clustering analyses using either PCA (principle component analysis) or information-theory-based t-SNE (t-distributed stochastic neighboring embedding) algorithm, we found that layer-specific distribution of dendritic terminals and planar projection directions are the most important type-specific attributes and they are sufficient to differentiate the Tm neurons. In sharp contrast, standard morphometric parameters, such as branch numbers and bifurcation topologies, are similar among these Tm neurons and these parameters are incapable of differentiating Tm neurons dendritic patterns. To determine the molecular mechanisms controlling dendritic patterning during development, we carried out genetic screens for morphological defects in Tm20 and Dm8 dendrites. We have identified adhesion receptors, morphogen receptors, signaling molecules, and cytoskeletal regulators that are cell-autonomously required in Tm20 or Dm8 neurons for proper dendritic development. In particular, the classical cadherin N-cadherin is required in Tm20 neurons for layer-specific initiation of main dendritic branching points. Unlike wild-type Tm20 neurons, which extended most dendritic branches from one or two primary branching nodes located in the medulla M3 layer, Ncad mutant Tm20 neurons shifted the main dendritic nodes to the M2 layers. The layer shift of the main branching nodes in Ncad mutant Tm20 correlates with an alteration of layer-specific targeting of their dendritic arbors and to some degree, their planar projection direction. Interestingly, the total dendritic length was unaffected, suggesting that Ncad mutation specifically affects the initiation of primary dendritic branches rather than branch trimming. We identified two pathways that regulate the sizes of dendritic trees. We found that the TGF-beta/Activin signaling pathway negatively controls the sizes of the dendritic fields of Tm20 and Dm8. Mutant Tm20 lacking Activin signaling components, such as the receptor Baboon and the downstream transcription factor Smad2, elaborated an expanded dendritic tree, spanning several medulla columns. Morphometric analyses based on a Kaplan-Meier non-parametric estimator further showed that baboon and smad2 mutations significantly reduced dendritic termination frequency. Using a modified GRASP method, we found that the expanded dendritic tree of mutant Tm20 forms aberrant synaptic contacts with several neighboring R8 photoreceptors. In contrast, wild-type Tm20 forms synaptic connections with single R8 photoreceptors in its cognate column. RNAi-mediated knockdown of Activin in R7s and R8s caused an abnormal expansion of dendritic fields of Dm8 and Tm20, respectively. These results indicate that photoreceptors R7 and R8 provide Activin specifically for their respective synaptic targets, Dm8 and Tm20. We found that the insulin signaling pathway positively regulates the dendritic tree size of Dm8 but not Tm20 neurons. Mutant Dm8 neurons lacking insulin receptor or the downstream signaling components TOR (target of rapamycin) or Rheb have a small dendritic tree while mutant Dm8 neurons lacking the negative regulator of the insulin signaling pathway, Pten (Phosphatase and tensin homolog) or TSC1 (Tuberous Sclerosis 1), have an expanded dendritic tree, as compared to the wild-type. Mutations in TOR regulatory genes, such as TSC1/2 and AKT, have been associated with several focal malformations of cortical development (MCD) subtypes associated with epilepsy, collectively called mTORopathies. The Dm8 system we developed allows the dissections of the complex phenotypes of the TOR pathway at the single-cell resolution and the segregation of developmental and compensatory effects.
