An imaging analysis protocol to trace, quantify, and model multi-signal neuron morphology

Iyer

et al. 2023

Preprint

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Dendrites are the primary points of sensory or synaptic inputs to a neuron and play an essential role in synaptic integration and neural function. Despite the functional importance of dendrites, relatively less is known about the underlying mechanisms regulating cell-type specific dendritic patterning. Herein, we have dissected functional roles of a previously uncharacterized gene,CG3995, in cell-type specific dendritic development inDrosophila melanogaster.CG3995, which we have namedbedwarfed(bdwf), encodes a zinc-finger BED-type protein which is required for proportional growth and branching of dendritic arbors, exhibits nucleocytoplasmic expression, and functions in both transcriptional and translational cellular pathways. At the transcriptional level, we demonstrate a reciprocal regulatory relationship between Bdwf and the homeodomain transcription factor (TF) Cut. We show that Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins produced phenotypically similar dendritic hypotrophy defects as observed in bdwf mutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, our findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. Taken together, these results provide new insights into the complex, combinatorial and multi-functional roles of transcription factors (TFs) in directing diversification of cell-type specific dendritic development.

Section: Resultsmentioning

confidence: 99%

The Zinc-BED transcription factor Bedwarfed promotes proportional dendritic growth and branching through transcriptional and translational regulation inDrosophila

Iyer

et al. 2023

Preprint

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“…Multi-signal morphological reconstructions were acquired using previously described methods (Nanda et al, 2021(Nanda et al, , 2018a, as briefly summarized below. The processed data have been deposited in Mendeley.com along with the analysis and modeling code for open-access release upon publication of this manuscript (preview: data.mendeley.com/datasets/cj69j8bpn8/draft?a=b9d99a91-9048-4974-b946-f99c397a79ec).…”

Section: Methodsmentioning

confidence: 99%

Local microtubule and F-actin distributions fully determine the spatial geometry ofDrosophilasensory dendritic arbors

Nanda

Cox

et al. 2023

Preprint

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Dendritic morphology underlies the source and processing of neuronal signal inputs. Morphology can be broadly described by two types of geometric characteristics. The first is dendrogram topology, defined by the length and frequency of the arbor branches; the second is spatial embedding, mainly determined by branch angles and tortuosity. We have previously demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. Here we relate the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. We first reconstruct and analyze 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes. We observe that branches with higher microtubule concentration are overall straighter and tend to deviate less from the direction of their parent branch. F-actin displays a similar effect on the angular deviation from the parent branch direction, but its influence on branch tortuosity varies by class and genotype. We then create a computational model of dendritic morphology purely constrained by the cytoskeletal composition imaged from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors.

“…Multichannel cytoskeletal reconstructions and quantitative analyses were performed using a previously described method ( Nanda et al, 2021 ). MT or F-actin quantity of a compartment is defined as:…”

Section: Methodsmentioning

confidence: 99%

PP2A phosphatase regulates cell-type specific cytoskeletal organization to drive dendrite diversity

Lottes

Nanda

et al. 2022

Front. Mol. Neurosci.

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Uncovering molecular mechanisms regulating dendritic diversification is essential to understanding the formation and modulation of functional neural circuitry. Transcription factors play critical roles in promoting dendritic diversity and here, we identify PP2A phosphatase function as a downstream effector of Cut-mediated transcriptional regulation of dendrite development. Mutant analyses of the PP2A catalytic subunit (mts) or the scaffolding subunit (PP2A-29B) reveal cell-type specific regulatory effects with the PP2A complex required to promote dendritic growth and branching in Drosophila Class IV (CIV) multidendritic (md) neurons, whereas in Class I (CI) md neurons, PP2A functions in restricting dendritic arborization. Cytoskeletal analyses reveal requirements for Mts in regulating microtubule stability/polarity and F-actin organization/dynamics. In CIV neurons, mts knockdown leads to reductions in dendritic localization of organelles including mitochondria and satellite Golgi outposts, while CI neurons show increased Golgi outpost trafficking along the dendritic arbor. Further, mts mutant neurons exhibit defects in neuronal polarity/compartmentalization. Finally, genetic interaction analyses suggest β-tubulin subunit 85D is a common PP2A target in CI and CIV neurons, while FoxO is a putative target in CI neurons.