Descending neurons (DNs) occupy a key position in the sensorimotor hierarchy, conveying signals from the brain to the rest of the body below the neck. In Drosophila melanogaster flies, approximately 480 DN cell types have been described from electron-microscopy image datasets. Genetic access to these cell types is crucial for further investigation of their role in generating behaviour. We previously conducted the first large-scale survey of Drosophila melanogaster DNs, describing 98 unique cell types from light microscopy and generating cell-type-specific split-Gal4 driver lines for 65 of them. Here, we extend our previous work, describing the morphology of 137 additional DN types from light microscopy, bringing the total number DN types identified in light microscopy datasets to 235, or nearly 50%. In addition, we produced 500 new sparse split-Gal4 driver lines and compiled a list of previously published DN lines from the literature for a combined list of 738 split-Gal4 driver lines targeting 171 DN types.

Simultaneous recordings from hundreds or thousands of neurons are becoming routine because of innovations in instrumentation, molecular tools, and data processing software. Such recordings can be analyzed with data science methods, but it is not immediately clear what methods to use or how to adapt them for neuroscience applications. We review, categorize, and illustrate diverse analysis methods for neural population recordings and describe how these methods have been used to make progress on longstanding questions in neuroscience. We review a variety of approaches, ranging from the mathematically simple to the complex, from exploratory to hypothesis-driven, and from recently developed to more established methods. We also illustrate some of the common statistical pitfalls in analyzing large-scale neural data.

The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.

e13028Background: Liquid biopsy has emerged as a powerful, minimally invasive tool for predicting treatment response and survival in breast and other advanced cancers. However, the detection and characterization of circulating tumor cells (CTCs) — a key factor in metastatic progression—remain challenging due to their low frequency and reliance on manual, time-intensive validation using only a couple of established methods for immunofluorescence staining, such as CellSearch. Harnessing deep learning for automated CTC detection and characterization of the blood cells interacting with CTCs holds the potential to advance prognostic evaluations and guide more effective therapies significantly. Methods: Leveraging FDA-approved CellSearch technology and sequencing approaches, we analyzed 2,853 blood specimens, longitudinally collected from 1358 patients with advanced cancer (breast, prostate, etc) and additional diseases. We built a novel deep learning platform, CTCpose, which integrates machine learning and AI-driven image analysis to automate the detection and categorization of CTCs, white blood cells (WBCs), and their clustering interactions. We extracted cellular and nuclear features to enable precise evaluation of individual CTCs, WBCs, homotypic CTC clusters, heterotypic CTC–WBC clusters, and immune cell aggregates. Results: By employing the CTCpose platform, we achieved fully automated identification of CTCs and immune cells, unraveling the spatial organization and functional characteristics of both homotypic and heterotypic clusters. These highly granular assessments revealed clinically significant correlations with patient survival, disease progression, and therapeutic outcomes. Our data underscore the critical role of CTC–immune cell interactions and the dynamic shifts in CTC phenotypes—both as single cells and clusters—in stratifying patients by risk and informing treatment strategies. Conclusions: This work illustrates the transformative power of deep learning in the analysis of liquid biopsy samples. By overcoming the limitations of traditional CTC detection, we have established a robust framework that integrates imaging data with large-scale patient cohorts to deliver predictive models of high clinical relevance. The CTCpose platform not only refines our understanding of CTC–immune cell biology but also paves the way for personalized oncology approaches, highlighting the impactful convergence of artificial intelligence and precision medicine.

Regulation of transcription during embryogenesis is key to development and differentiation. To study transcript expression throughout Caenorhabditis elegans embryogenesis at single-molecule resolution, we developed a high-throughput single-molecule fluorescence in situ hybridization (smFISH) method that relies on computational methods to developmentally stage embryos and quantify individual mRNA molecules in single embryos. We applied our system to sdc-2, a zygotically transcribed gene essential for hermaphrodite development and dosage compensation. We found that sdc-2 is rapidly activated during early embryogenesis by increasing both the number of mRNAs produced per transcription site and the frequency of sites engaged in transcription. Knockdown of sdc-2 and dpy-27, a subunit of the dosage compensation complex (DCC), increased the number of active transcription sites for the X chromosomal gene dpy-23 but not the autosomal gene mdh-1, suggesting that the DCC reduces the frequency of dpy-23 transcription. The temporal resolution from in silico staging of embryos showed that the deletion of a single DCC recruitment element near the dpy-23 gene causes higher dpy-23 mRNA expression after the start of dosage compensation, which could not be resolved using mRNAseq from mixed-stage embryos. In summary, we have established a computational approach to quantify temporal regulation of transcription throughout C. elegans embryogenesis and demonstrated its potential to provide new insights into developmental gene regulation.

Regulation of transcription during embryogenesis is key to development and differentiation. To study transcript expression throughout Caenorhabditis elegans embryogenesis at single-molecule resolution, we developed a high-throughput single-molecule fluorescence in situ hybridization (smFISH) method that relies on computational methods to developmentally stage embryos and quantify individual mRNA molecules in single embryos. We applied our system to sdc-2, a zygotically transcribed gene essential for hermaphrodite development and dosage compensation. We found that sdc-2 is rapidly activated during early embryogenesis by increasing both the number of mRNAs produced per transcription site and the frequency of sites engaged in transcription. Knockdown of sdc-2 and dpy-27, a subunit of the dosage compensation complex (DCC), increased the number of active transcription sites for the X chromosomal gene dpy-23 but not the autosomal gene mdh-1, suggesting that the DCC reduces the frequency of dpy-23 transcription. The temporal resolution from in silico staging of embryos showed that the deletion of a single DCC recruitment element near the dpy-23 gene causes higher dpy-23 mRNA expression after the start of dosage compensation, which could not be resolved using mRNAseq from mixed-stage embryos. In summary, we have established a computational approach to quantify temporal regulation of transcription throughout C. elegans embryogenesis and demonstrated its potential to provide new insights into developmental gene regulation.Competing Interest StatementThe authors have declared no competing interest.

Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cisTEM image processing packages, to focus classify on specific regions of a particle and detect potential covariance. The strategies are based on masking the region of interest using either a 2-D mask applied to reference projections and particle images, or a 3-D mask applied to the 3-D volume. We show that focused classification approaches can be used to study structural covariance, a concept that is likely to gain more importance as datasets grow in size, allowing the distinction of more structural states and smaller differences between states. Finally, we apply the approaches to an experimental dataset containing the HIV-1 Transactivation Response (TAR) element RNA fused into the large bacterial ribosomal subunit to deconvolve structural mobility within localized regions of interest, and to a dataset containing assembly intermediates of the large subunit to measure structural covariance.

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

The ability of fluorescence microscopy to simultaneously image multiple specific molecules of interest has allowed biologists to infer macromolecular organization and colocalization in fixed and live samples. However, a number of factors could affect these analyses, and colocalization is a misnomer. We propose that image similarity coefficient as a better and more descriptive term. In this chapter we will discuss many of the factors involved with determining image similarity including our perception of color in images. In addition, the correct use of several commonly accepted methods such as Pearson’s correlation coefficient, Manders’ overlap coefficient, and Spearman’s ranked correlation coefficient is discussed.

Chromosome inversions are of unique importance in the evolution of genomes and species because when heterozygous with a standard arrangement chromosome, they suppress meiotic crossovers within the inversion. In Drosophila species, heterozygous inversions also cause the interchromosomal effect, whereby the presence of a heterozygous inversion induces a dramatic increase in crossover frequencies in the remainder of the genome within a single meiosis. To date, the interchromosomal effect has been studied exclusively in species that also have high frequencies of inversions in wild populations. We took advantage of a recently developed approach for generating inversions in Drosophila simulans, a species that does not have inversions in wild populations, to ask if there is an interchromosomal effect. We used the existing chromosome 3R balancer and generated a new chromosome 2L balancer to assay for the interchromosomal effect genetically and cytologically. We found no evidence of an interchromosomal effect in D. simulans. To gain insight into the underlying mechanistic reasons, we qualitatively analyzed the relationship between meiotic double-strand break formation and synaptonemal complex assembly. We find that the synaptonemal complex is assembled prior to double-strand break formation as in D. melanogaster; however, we show that the synaptonemal complex is assembled prior to localization of the oocyte determination factor Orb, whereas in D. melanogaster, synaptonemal complex formation does not begin until Orb is localized. Together, our data show heterozygous inversions in D. simulans do not induce an interchromosomal effect and that there are differences in the developmental programming of the early stages of meiosis.