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18The generation of neuronal diversity is essential for circuit formation and behavior. Morphological 19 differences in sequentially born neurons could be due to intrinsic molecular identity specified by 20 temporal transcription factors (henceforth called intrinsic temporal identity) or due to changing 21 extrinsic cues. Here we use the Drosophila NB7-1 lineage to address this question. NB7-1 22 sequentially generates the U1-U5 motor neurons; each has a distinct intrinsic temporal identity due 23 to inheritance of a different temporal transcription factor at time of birth. Here we show that the U1-24 U5 neurons project axons sequentially, followed by sequential dendrite extension. We misexpress 25 the earliest temporal transcription factor, Hunchback, to create "ectopic" U1 neurons with an early 26 intrinsic temporal identity but later birth-order. These ectopic U1 neurons have axon muscle 27 targeting and dendrite neuropil targeting consistent with U1 intrinsic temporal identity, rather than 28 their time of birth or differentiation. We conclude that intrinsic temporal identity plays a major role in 29 establishing both motor axon muscle targeting and dendritic arbor targeting, which are required for 30 proper motor circuit development. 31 32 33 Introduction 34 35 Axon and dendrite targeting is an essential step in neural circuit formation, and may even be 36 sufficient for proper connectivity in some cases, as postulated in Peters' Rule (Peters and 37 Feldman, 1976; Rees et al., 2017; Stepanyants and Chklovskii, 2005). In both Drosophila and 38 mammals, individual progenitors generate a series of neurons that differ in axon and dendrite 39 targeting (Doe, 2017; Kohwi and Doe, 2013; Li et al., 2013a; Pearson and Doe, 2004; Rossi et al., 40 2016). In all examples, neurons born at different times have intrinsic molecular differences due to 41 temporal transcription factors (TTFs) present at their time of birth (reviewed in Kohwi and Doe, 42 2013), which could specify neuronal morphology. Conversely, there are likely changing extrinsic 43 cues present at the time of neuronal differentiation that could also influence neuronal morphology, 44 such as modulation of global pathfinding cues or addition of axon and dendrite processes 45 throughout neurogenesis. Teasing out the relative contributions of intrinsic or extrinsic factors 46 requires heterochronic experiments where either intrinsic or extrinsic cues are altered to create a 47 mismatch, and the effects on axon and dendrite targeting are assessed. 48 Several experiments highlight the importance of extrinsic cues present at the time of 49 neuronal differentiation in establishing axon or dendrite targeting. For example, transplantation 50 of rat fetal occipital cortical tissue into the rostral cortex of a more developmentally mature 51 newborn host results in axonal projections characteristic of the host site (O'Leary and 52 Stanfield, 1989; Schlaggar and O'Leary, 1991; Stanfield and O'Leary, 1985). Similarly, 53 transplantation of embryonic day 15 fetal occipital...

The generation of neuronal diversity is essential for circuit formation and behavior. Morphological differences in sequentially born neurons could be due to intrinsic molecular identity specified by temporal transcription factors (henceforth called intrinsic temporal identity) or due to changing extrinsic cues. Here, we have used the Drosophila NB7-1 lineage to address this issue. NB7-1 generates the U1-U5 motor neurons sequentially; each has a distinct intrinsic temporal identity due to inheritance of different temporal transcription factors at its time of birth. We show that the U1-U5 neurons project axons sequentially, followed by sequential dendrite extension. We misexpressed the earliest temporal transcription factor, Hunchback, to create 'ectopic' U1 neurons with an early intrinsic temporal identity but later birth-order. These ectopic U1 neurons have axon muscle targeting and dendrite neuropil targeting that are consistent with U1 intrinsic temporal identity, rather than with their time of birth or differentiation. We conclude that intrinsic temporal identity plays a major role in establishing both motor axon muscle targeting and dendritic arbor targeting, which are required for proper motor circuit development.