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 Drosophila type II neuroblast lineages present an attractive model to investigate the neurogenesis and differentiation process as they adapt to a process similar to that in the human outer subventricular zone. We perform targeted single-cell mRNA sequencing in third instar larval brains to study this process of the type II NB lineage. Combining prior knowledge, in silico analyses, and in situ validation, our multi-informatic investigation describes the molecular landscape from a single developmental snapshot. 17 markers are identified to differentiate distinct maturation stages. 30 markers are identified to specify the stem cell origin and/or cell division numbers of INPs, and at least 12 neuronal subtypes are identified. To foster future discoveries, we provide annotated tables of pairwise gene-gene correlation in single cells and MiCV, a web tool for interactively analyzing scRNA-seq datasets. Taken together, these resources advance our understanding of the neural differentiation process at the molecular level.

SUMMARY Elucidating cell lineages provides crucial understanding of development. Recently developed sequencing-based techniques enhance the scale of lineage tracing but eliminate the spatial information offered by conventional approaches. Multi-spectral labeling techniques, such as Brainbow, have the potential to identify lineage-related cells in situ. Here, we report nuclear Bitbow (nBitbow), a “digital” version of Brainbow that greatly expands the color diversity for scoring cells, and a suite of statistical methods for quantifying the lineage relationship of any two cells. Applying these tools to the Drosophila peripheral nervous system (PNS), we determined lineage relationship between all neuronal pairs. This study demonstrates nBitbow as an efficient tool for in situ lineage mapping, and the complete lineage relationship among larval PNS neurons opens new possibilities for studying how neurons gain specific features and circuit connectivity.

There is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury. However, the molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to significant pediatric lung diseases, indicating a need to uncover fetal programs promoting vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial endothelial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation with gene ontology and histological analysis allowed us to segregate the developing artery endothelium into functionally and spatially distinct novel subpopulations. Expression of Cxcl12 is highest in the distal arterial endothelial subpopulation, a compartment enriched in genes for vascular development. Accordingly, disruption of CXCL12 signaling led to not only abnormal branching but also distal vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.