Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism
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Evaluation Summary:
This manuscript describes a pipeline to assess the number of a certain cell type in the larval Drosophila brain. Their work provides automated neuronal segmentation and topographical analysis methods for the whole larval nervous system organization in flies, revealing the previously unexpected sexual dimorphism. This paper may be of interest to the large class of neuroscientists and specialists, from those who use larval Drosophila as their study model to others who are generally interested in connectomics and transcriptomics, among others.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of Caenorhabditis elegans . Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.
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Evaluation Summary:
This manuscript describes a pipeline to assess the number of a certain cell type in the larval Drosophila brain. Their work provides automated neuronal segmentation and topographical analysis methods for the whole larval nervous system organization in flies, revealing the previously unexpected sexual dimorphism. This paper may be of interest to the large class of neuroscientists and specialists, from those who use larval Drosophila as their study model to others who are generally interested in connectomics and transcriptomics, among others.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this study the authors develop a pipeline that combines molecular genetics, whole brain imaging and computational approaches to quantitate the precise numbers of larval-specific neurons and glia within the larval Drosophila brain.
To this end, they develop Gal4 driver lines that either specifically target functional neurons or glia, and a UAS-reporter line that specifically labels cell nuclei with little background. By stitching together multi view light sheet microscopy data of such labelled brains the authors generate a brain template from which they can now extract quantitative information. The first of these is cell number - they find that in the larval brain, the number of neurons is significantly less than was previously estimated and that of glia was significantly more. They also find that larval …
Reviewer #1 (Public Review):
In this study the authors develop a pipeline that combines molecular genetics, whole brain imaging and computational approaches to quantitate the precise numbers of larval-specific neurons and glia within the larval Drosophila brain.
To this end, they develop Gal4 driver lines that either specifically target functional neurons or glia, and a UAS-reporter line that specifically labels cell nuclei with little background. By stitching together multi view light sheet microscopy data of such labelled brains the authors generate a brain template from which they can now extract quantitative information. The first of these is cell number - they find that in the larval brain, the number of neurons is significantly less than was previously estimated and that of glia was significantly more. They also find that larval brains are sexually dimorphic - females have more neurons than males and males have more glia than females. They perform topological analyses to show that this difference in numbers may translate to subtle structural differences between male and female larval brains. Finally, they show that their approach can also be used to determine gene expression frequency.
In summary, this is a thorough study that provides surprising insights about cell numbers in the larval brain.
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Reviewer #2 (Public Review):
The current study investigated the topography of the larval central nervous system of Drosophila by combining newly established genetic tools, light-sheet microscopy, automated nuclei segmentation, and computational analyses for spatial nuclei organization. They found that the number of neurons counted by known synaptic marker expressions are stable within a gender but sexually dimorphic, i.e., females having more neurons than males. Furthermore, by exploiting elaborated computational techniques that analyze the geometrical features of neuronal nuclei coordinate, the authors revealed hitherto-unknown topographical features that differ by gender, further corroborating their conclusions. The current study provides a promising and powerful platform that facilitates the functional decomposition of the entire …
Reviewer #2 (Public Review):
The current study investigated the topography of the larval central nervous system of Drosophila by combining newly established genetic tools, light-sheet microscopy, automated nuclei segmentation, and computational analyses for spatial nuclei organization. They found that the number of neurons counted by known synaptic marker expressions are stable within a gender but sexually dimorphic, i.e., females having more neurons than males. Furthermore, by exploiting elaborated computational techniques that analyze the geometrical features of neuronal nuclei coordinate, the authors revealed hitherto-unknown topographical features that differ by gender, further corroborating their conclusions. The current study provides a promising and powerful platform that facilitates the functional decomposition of the entire nervous system of the model organism and complements the ongoing connectome project elsewhere.
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