Bitbow: a digital format of Brainbow enables highly efficient neuronal lineage tracing and morphology reconstruction in single brains

Abstract

10Identifying the cellular origins and mapping the dendritic and axonal arbors of neurons have 11 been century old quests to understand the heterogeneity among these brain cells. Classical 12 chemical and genetic methods take advantage of light microscopy and sparse labeling to 13 unambiguously, albeit inefficiently, trace a few neuronal lineages or reconstruct their 14 morphologies in each sampled brain. To improve the analysis throughput, we designed Bitbow, 15 a digital format of Brainbow which exponentially expands the color palette to provide tens of 16 thousands of spectrally resolved unique labels. We generated transgenic Bitbow Drosophila 17 lines, established statistical tools, and streamlined sample preparation, image processing and 18 data analysis pipelines to allow conveniently mapping neural lineages, studying neuronal 19 morphology and revealing neural network patterns with an unprecedented speed, scale and 20 resolution. 21 One way to generate more unique labels for lineage tracing is to localize the same FPs to 48 different subcellular compartments. In strategies such as CLoNe and MAGIC, Brainbow 49 cassettes targeted to cytoplasm, cell membrane, nucleus, and/or mitochondria were co-50 electroporated with transposase for genome integration, which allowed the differentiation of 51 neighboring progenies in chick and mouse embryos with fewer color collisions 26,27 . However, 52 the number of expression cassettes being integrated in each cell is random in these 53 experiments, leading to uncertainty in each color's appearance probability which complicates 54 quantitative analysis. The Raeppli strategy solves this problem by generating a transgenic 55 Drosophila which utilizes 4 FPs to create up to 4 x 4 = 16 membrane and nucleus color 56 combinations 16 . In parallel, strategies such as TIE-DYE and MultiColor FlpOut (MCFO) attempt to 57 generate more color combinations by stochastically removing the expression stops from each 58 3 FP module 15,28 . While inserting 3 different modules into 3 genomic loci allows generating up to 59 2 3 -1=7 unique labels, it is difficult to insert more modules to more genomic loci in a single 60 transgenic animal. 61 Here we present Bitbow, a digital format of Brainbow to greatly expand the unique color 62 pool from a single transgenic cassette. Unlike the original Brainbow, whose FP choices are 63 exclusive in one cassette, Bitbow allows each FP to independently express in an ON or OFF state 64 upon recombination. Color coding by each FP's binary status is similar to the information coding 65 by each bit in computer memory, thus leading to the name Bitbow. In a recent study, we 66 implemented the Bitbow1 design to target 5 spectrally distinct FPs to the nucleus for lineage 67 tracing 33 . Here, we present novel Bitbow1 flies which encode up to 32,767 unique "colors" 68 (Bitbow codes) in a single transgenic animal. This allows reliable lineage tracing without 69 complicated statistical tests 33 . To better enable morphology tracing, we generated Bitbow2, 70 which...

The molecular landscape of neural differentiation in the developing Drosophila brain revealed by targeted scRNA-seq and multi-informatic analysis

“…Genetic constructs based around CRISPR-Cas9 (Raj et al, 2017;Spanjaard et al, 2018) and the Cre/Lox system (Kalhor et al, 2018;Pei et al, 2017;Weber et al, 2016) have been developed for this purpose, although which exact lineage was labeled by a particular barcode was still unknown. The introduction of a spectrally unique barcode for each neuroblast lineage, in a similar vein to the recently developed Bitbow lineage tracking strategy (Li et al, 2020;Veling et al, 2019), would be advantageous as they can provide direct in situ evidence for neuroblast lineage identity.…”

nGauge: Integrated and extensible neuron morphology analysis in Python

“…Combined with visualization tools, nGauge empowers the creation of publication-quality figures with ease. In fact, during the development of the nGauge project, we have already applied all of the individual modules to produce results both in publication and in preparation, finding it to be a very effective toolkit for efficient data science (Shen et al, 2020;Li et al, 2020;Dizaji et al, 2020;Duan et al, 2020).…”

“…Each square is normalized to the volume of the cell identified in the X-axis, which is depicted in the bar plot to the bottom of the figure. (Li et al, 2020) is plotted atop the raw data. A full animation of this figure is available as Movie S2 in (Li et al, 2020).…”

The molecular landscape of neural differentiation in the developingDrosophilabrain revealed by targeted scRNA-seq and a multi-informatic analysis paradigm

“…The introduction of a spectrally unique barcode for each neuroblast lineage, in a similar vein to the recently developed Bitbow lineage tracking strategy (Li et al, 2020;Veling et al, 2019), would be advantageous as they can provide direct in situ evidence for neuroblast lineage identity.…”