Mechanisms of neural patterning and the generation of neural diversity in the brain. We have shown that neural diversity in the optic lobe of Drosophila is generated by three mechanisms: Each neural stem cell (neuroblast; NB) expresses sequentially a series of temporal transcription factors (tTF) and divides to produce an intermediate precursor that divides once more to generate a NotchON and a NotchOFF neuron. The neuroepithelium that generates NBs is patterned by spatial TFs (sTFs) and by signaling molecules. Different regions of the neuroepithelium generate NBs that undergo the same tTF series but produce different neurons due to spatial patterning of the neuroepithelium of origin. We offer to continue our investigations into the development of the medulla and address whether stochastic factors complement the deterministic and intrinsic programs that generate diversity (Aim 1) and investigate how many different spatial regions comprise the NE and how this spatial patterning controls the stoichiometry of neurons (Aim 2). There are many more than 800 NBs to produce neurons populating the 800 medulla columns that process visual information from the 800 unit-eyes in the fly retina. We will test whether an excess number of the same neurons is produced from all the NBs and culled later by apoptosis, or whether the temporal series progresses independently of cell cycle: In the latter case, the lack of coordination between the progression of the temporal series and cell cycle introduces a stochastic component that would allow flexibility in the identity (and number) of the neurons produced by each NB. The role of stochastic patterning of neural stem cells has been demonstrated in mammals but is very poorly understood. The repetitive structure and precise genetic manipulations in the fly optic lobe make this system ideal to address these questions: Aim 1: Progression of the temporal series and cell cycle: The many NBs (at least twice as many as columns) suggest that neurons are produced in excess and culled, or that each NB only produces stochastically a subset of the neurons. Aim 1.1: Do NBs produce excess numbers of neurons and those that do not find a partner are later culled by cell death? Aim 1.2: Is the progression of temporal windows coordinated with cell cycle? Aim 1.3: Are all temporal windows used to generate all the neurons that a given NB could produce? Aim 1.4: What is the temporal window of origin of the ~100 medulla neurons? Aim 1.5: Does the length of temporal windows determine the number of neurons produced at each temporal window? Aim 1.6: Does the speed of transition of temporal windows rely on regulation of RNA stability and/or translation status? Aim 2: Spatial patterning and epigenetic memory: How do NBs from different spatial domains undergo the same temporal series but produce different neurons in different stoichiometry? Aim 2.1: How does Dpp signaling generate at least three subdomains within one of the spatial domains? Ai...