multiplex-detection-gene-expression-intact-drosophila-brain-using

Multiplex Detection of Gene Expression in the Intact <em>Drosophila</em> Brain Using Expansion-Assisted Iterative Fluorescence <em>In Situ</em> Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

Howard Hughes Medical Institute

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.

A complete developmental sequence of a Drosophila neuronal lineage as revealed by twin-spot MARCM.

The anterodorsal projection neuron lineage of Drosophila melanogaster produces 40 neuronal types in a stereotypic order. Here we take advantage of this complete lineage sequence to examine the role of known temporal fating factors, including Chinmo and the Hb/Kr/Pdm/Cas transcriptional cascade, within this diverse central brain lineage. Kr mutation affects the temporal fate of the neuroblast (NB) itself, causing a single fate to be skipped, whereas Chinmo null only elicits fate transformation of NB progeny without altering cell counts. Notably, Chinmo operates in two separate windows to prevent fate transformation (into the subsequent Chinmo-indenpendent fate) within each window. By contrast, Hb/Pdm/Cas play no detectable role, indicating that Kr either acts outside of the cascade identified in the ventral nerve cord or that redundancy exists at the level of fating factors. Therefore, hierarchical fating mechanisms operate within the lineage to generate neuronal diversity in an unprecedented fashion.

Hierarchical deployment of factors regulating temporal fate in a diverse neuronal lineage of the Drosophila central brain.

The insect brain can be divided into neuropils that are formed by neurites of both local and remote origin. The complexity of the interconnections obscures how these neuropils are established and interconnected through development. The Drosophila central brain develops from a fixed number of neuroblasts (NBs) that deposit neurons in regional clusters.

Clonal development and organization of the adult Drosophila central brain.

The Drosophila central brain develops from a fixed number of neuroblasts. Each neuroblast makes a clone of neurons that exhibit common trajectories. Here we identified 15 distinct clones that carry larval-born neurons innervating the Drosophila central complex (CX), which consists of four midline structures including the protocerebral bridge (PB), fan-shaped body (FB), ellipsoid body (EB), and noduli (NO). Clonal analysis revealed that the small-field CX neurons, which establish intricate projections across different CX substructures, exist in four isomorphic groups that respectively derive from four complex posterior asense-negative lineages. In terms of the region-characteristic large-field CX neurons, we found that two lineages make PB neurons, 10 lineages produce FB neurons, three lineages generate EB neurons, and two lineages yield NO neurons. The diverse FB developmental origins reflect the discrete input pathways for different FB subcompartments. Clonal analysis enlightens both development and anatomy of the insect locomotor control center.

Diverse neuronal lineages make stereotyped contributions to the Drosophila locomotor control center, the central complex.

The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.

Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts.

Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, we profiled type II neuroblasts' transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, we reveal that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. We therefore propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.

Stem Cell-Intrinsic, Seven-up-Triggered Temporal Factor Gradients Diversify Intermediate Neural Progenitors.

Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells.  central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18  central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.

Conservation and divergence of related neuronal lineages in the  central brain.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of  neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

A searchable image resource of  GAL4 driver expression patterns with single neuron resolution.

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system (CNS) have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in  and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult  CNS and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell-type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

Article	Year
A complete developmental sequence of a Drosophila neuronal lineage as revealed by twin-spot MARCM. PLoS biology\| PubMed ID: 20808769	2010
Hierarchical deployment of factors regulating temporal fate in a diverse neuronal lineage of the Drosophila central brain. Neuron\| PubMed ID: 22365543	2012
Clonal development and organization of the adult Drosophila central brain. Current biology : CB\| PubMed ID: 23541733	2013
Diverse neuronal lineages make stereotyped contributions to the Drosophila locomotor control center, the central complex. The Journal of comparative neurology\| PubMed ID: 23696496	2013
Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts. Nature neuroscience\| PubMed ID: 24561995	2014
Stem Cell-Intrinsic, Seven-up-Triggered Temporal Factor Gradients Diversify Intermediate Neural Progenitors. Current biology : CB\| PubMed ID: 28434858	2017
Conservation and divergence of related neuronal lineages in the central brain. eLife\| PubMed ID: 32255422	2020
A searchable image resource of GAL4 driver expression patterns with single neuron resolution. eLife\| PubMed ID: 36820523	2023
A split-GAL4 driver line resource for neuron types. eLife\| PubMed ID: 39854223	2025

Yisheng He

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