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bioRxiv2024

Temporal Microbiome Changes in Axolotl Limb Regeneration: Stage-Specific Restructuring of Bacterial and Fungal Communities with a Flavobacterium Bloom During Blastema Proliferation

The intricate relationship between regeneration and microbiota has recently gained attention, spanning diverse model organisms. Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite its significance, a noticeable gap exists in understanding the interplay between axolotl regeneration and its microbiome. Here, we analyze in depth bacterial 16S rRNA amplicon dataset that we reported before as data resource and profile fungal community by sequencing ITS amplicons at the critical stages of limb regeneration (0-1-4-7-30-60 days post amputation, "dpa"). Results reveal a decline in richness and evenness in the course of limb regeneration, with bacterial community richness recovering beyond 30 dpa unlike fungi community. Beta diversity analysis reveals precise restructuring of the bacterial community along the three phases of limb regeneration, contrasting with less congruent changes in the fungal community. Temporal dynamics of the bacterial community highlight prevalent anaerobic bacteria in initiation phase and Flavobacterium bloom in the early phase correlating with limb blastema proliferation. Predicted functional analysis mirrors these shifts, emphasizing a transition from amino acid metabolism to lipid metabolism control. Fungal communities shift from Blastomycota to Ascomycota dominance in the late regeneration stage. Our findings provide ecologically relevant insights into stage specific role of microbiome contributions to axolotl limb regeneration.

microbiology↗

bioRxiv2024

Unravelling the Transcriptomic Symphony of Sarcopenia: Key Pathways and Hub Genes Altered by Muscle Ageing and Caloric Restriction Revealed by RNA Sequencing

Sarcopenia is a disease involving extensive loss of muscle mass and strength with age and is a major cause of disability and accidents in the elderly. Mechanisms purported to be involved in muscle ageing and sarcopenia are numerous but poorly understood, necessitating deeper study. Hence, we employed high-throughput RNA sequencing to explicate the global changes in protein-coding gene expression occurring in skeletal muscle with age. Caloric restriction (CR) is a proven prophylactic intervention against sarcopenia. Therefore, total RNA was isolated from the muscle tissue of both rats fed ad libitum and CR rats. Collected data were subjected to Gene Ontology, pathway, co-expression, and interaction network analyses. This revealed the functional pathways most activated by both ageing and CR, as well as the key "hub" proteins involved in their activation. RNA-seq revealed 442 protein-coding genes to be upregulated and 377 to be downregulated in aged muscle, compared to young muscle. Upregulated genes were commonly involved in protein folding and the immune response; meanwhile, downregulated genes were often related to developmental biology. CR was found to suppress 69.7% and rescue 57.8% of the genes found to be upregulated and downregulated in aged muscle, respectively. In addition, CR uniquely upregulated 291 and downregulated 304 protein-coding genes. Hub genes implicated in both ageing and CR included Gc, Plg, Irf7, Ifit3, Usp18, Rsad2, Blm and RT1-A2, whilst those exclusively implicated in CR responses included Alb, Apoa1, Ambp, F2, Apoh, Orm1, Mx1, Oasl2 and Rtp4. Hub genes involved in ageing but unaffected by CR included Fgg, Fga, Fgb and Serpinc1. In conclusion, this comprehensive RNA sequencing study highlighted gene expression patterns, hub genes and signalling pathways most affected by ageing in skeletal muscle. This data may provide the initial evidence for several targets for therapeutic interventions against sarcopenia.

genomics↗

bioRxiv2024

Insights into Cellular Evolution: Temporal Deep Learning Models and Analysis for Cell Image Classification

I.AO_SCPLOWBSTRACTC_SCPLOWUnderstanding the temporal evolution of cells poses a significant challenge in developmental biology. This study embarks on a comparative analysis of various machine-learning techniques to classify sequences of cell colony images, thereby aiming to capture dynamic transitions of cellular states. Utilizing transfer learning with advanced classification networks, we achieved high accuracy in single-timestamp image categorization. We introduce temporal models--LSTM, R-Transformer, and ViViT--to explore the effectiveness of integrating temporal features in classification, comparing their performance against non-temporal models. This research benchmarks various machine learning approaches in understanding cellular dynamics, setting a foundation for future studies to enhance our understanding of cellular developments with computational methods, contributing significantly to biological research advancements.

cell biology↗

bioRxiv2024

In utero adeno-associated virus (AAV)-mediated gene delivery targeting sensory and supporting cells in the embryonic mouse inner ear

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.

neuroscience↗

bioRxiv2024

Y chromosome shredding in Anopheles gambiae: insight into the cellular dynamics of a novel synthetic sex ratio distorter.

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species. Author summaryGenetic elements known as sex ratio meiotic drive can manipulate the sex ratio of offspring, favouring the male or female sex. This fascinating phenomenon has inspired the development of synthetic sex ratio distorter systems in several organisms. In species where females and males harbour XX and XY sex chromosomes respectively, the X-chromosome can be shredded during male gametogenesis, leading to the production of non-functional X-bearing sperm, while Y-bearing sperm are left intact and able to fertilise the eggs. These systems can produce offspring that are extremely biased towards males, which can be used as genetic tools to control harmful insect populations. In our study, we applied this molecular strategy to target the Y chromosome of Anopheles gambiae. Our aim was to investigate the cellular consequences of the shredding of this chromosome, the impact on meiosis and sperm selection, and the potential to achieve strong female bias in the offspring. The outcome of this study enhances our understanding of the molecular and biological mechanisms behind synthetic sex-ratio distorters in Anopheles mosquitoes, which could inform the development of vector control strategies that target sex ratio. Additionally, we present a genetic sexing strain able to produce mostly females, providing a valuable genetic tool for fundamental studies on this deadly vector.

molecular biology↗

bioRxiv2024

Unveiling Tissue Structure and Tumor Microenvironment from Spatially Resolved Transcriptomics by Hypergraph Learning

Spatial omics technologies have revolutionized life sciences by enabling the simultaneous acquisition of biomolecular and spatial information. Identifying spatial patterns is crucial for understanding organ development and tumor microenvironments. However, the emergence of diverse spatial omics resolutions in these technologies has made it challenging to accurately characterize spatial domains at finer resolutions. To address this, we propose HyperSTAR, a hypergraph-based method designed to precisely identify spatial domains across varying resolutions by leveraging higher-order relationships among spatially adjacent tissue programs. Specifically, a gene expression-guided hyperedge decomposition module is introduced to refine the hypergraph structure to accurately delineate spatial domains boundaries. Additionally, a hypergraph attention convolutional neural network is designed to adaptively learn the importance of each hyperedge, enhancing the models ability to capture complex higher-order relationships within spatially neighboring multi-spots and/or single cells. HyperSTAR outperforms existing graph neural network models in tasks such as uncovering tissue substructures, inferring spatiotemporal patterns, and denoising spatially resolved gene expressions. It effectively handles diverse spatial omics data types and scales seamlessly to large datasets. The method successfully reveals spatial heterogeneity in breast cancer sections, with findings validated through functional and survival analyses of independent clinical data. HyperSTAR represents a significant advancement in spatial omics analysis, representing a robust tool for exploring complex spatial patterns across varying resolutions and data types. Its ability to capture intricate higher-order relationships among spatially neighboring spots/cells makes it an invaluable tool for advancing research in life sciences, particularly in cancer and developmental biology.

bioinformatics↗

bioRxiv2024

A novel expression domain of extradenticle underlies the evolutionary developmental origin of the chelicerate patella

Neofunctionalization of duplicated gene copies is thought to be an important process underlying the origin of evolutionary novelty and provides an elegant mechanism for the origin of new phenotypic traits. One putative case where a new gene copy has been linked to a novel morphological trait is the origin of the arachnid patella, a taxonomically restricted leg segment. In spiders, the origin of this segment has been linked to the origin of the paralog dachshund-2, suggesting that a new gene facilitated the expression of a new trait. However, various arachnid groups that possess patellae do not have a copy of dachshund-2, disfavoring the direct link between gene origin and trait origin. We investigated the developmental genetic basis for patellar patterning in the harvestman Phalangium opilio, which lacks dachshund-2. Here, we show that the harvestman patella is established by a novel expression domain of the transcription factor extradenticle. Leveraging this definition of patellar identity, we surveyed targeted groups across chelicerate phylogeny to assess when this trait evolved. We show that a patellar homolog is present in Pycnogonida (sea spiders) and various arachnid orders, suggesting a single origin of the patella in the ancestor of Chelicerata. A potential loss of the patella is observed in Ixodida. Our results suggest that the modification of an ancient gene, rather than the neofunctionalization of a new gene copy, underlies the origin of the patella. Broadly, this work underscores the value of comparative data and broad taxonomic sampling when testing hypotheses in evolutionary developmental biology.

evolutionary biology↗

bioRxiv2024

Pioneer factor ETV2 safeguards endothelial cell specification by recruiting the repressor REST to restrict alternative lineage commitment

Mechanisms of cell fate specification remain a central question for developmental biology and regenerative medicine. The pioneer factor ETV2 is a master regulator for the endothelial cell (EC) lineage specification. Here, we studied mechanisms of ETV2-driven fate specification using a highly efficient system in which ETV2 directs human induced pluripotent stem cell-derived mesodermal progenitors to form ECs over two days. By applying CUT&RUN, single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses, we characterized the transcriptomic profiles, chromatin landscapes, dynamic cis-regulatory elements (CREs), and molecular features of EC cell differentiation mediated by ETV2. This defined the scope of ETV2 pioneering activity and identified its direct downstream target genes. Induced ETV2 expression both directed specification of endothelial progenitors and suppressed acquisition of alternative fates. Functional screening and candidate validation revealed cofactors essential for efficient EC specification, including the transcriptional activator GABPA. Surprisingly, the transcriptional repressor REST was also necessary for efficient EC specification. ETV2 recruited REST to occupy and repress non-EC lineage genes. Collectively, our study provides an unparalleled molecular analysis of EC specification at single-cell resolution and identifies the important role of pioneer factors to recruit repressors that suppress commitment to alternative lineages.

cell biology↗

bioRxiv2024

The genetic basis of the black pupae phenotype in tephritid fruit flies

The remarkable diversity of insect pigmentation offers a captivating avenue for exploring evolution and genetics. In tephritid fruit flies, decoding the molecular pathways underlying pigmentation traits also plays a central role in applied entomology. Mutant phenotypes like the black pupae (bp) have long been used as a component of genetic sexing strains, allowing male-only release in tephritid sterile insect technique applications. However, the genetic basis of bp remains largely unknown. Here, we present independent evidence from classical and modern genetics showing that the bp phenotype in the GUA10 strain of the Mexican fruit fly, Anastrepha ludens, is caused by a large deletion at the ebony locus resulting in the removal of the entire protein-coding region of the gene. Targeted knockout of ebony induced analogous bp phenotypes across six tephritid species spanning over 50 million years of divergent evolution. This functionally validated our findings and allowed for a deeper investigation into the role of Ebony in pigmentation and development in these species. Our study offers fundamental knowledge for developing new sexing strains based on the bp marker and for future evolutionary developmental biology studies in tephritid fruit flies.

genomics↗

bioRxiv2024

Promoting Human Intestinal Organoid Formation and Stimulation Using Piezoelectric Nanofiber Matrices

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology three days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.

bioengineering↗

bioRxiv2024

Exploring DNA movement through the application of droplet based high efficient chromatin conformation capture (DropHiChew) and loop velocity

This study presents a novel approach to understanding DNA movement dynamics through the development of a droplet-based, high-efficient chromatin conformation capture method, known as DropHiChew, and a new algorithm, loop velocity. DropHiChew, a user-friendly and cost-effective technique, employs the 10X single-cell systems allowing for easy experimental implementation. The loop velocity algorithm, on the other hand, enables the estimation of the speed and direction of cell development, providing a dynamic perspective on chromatin movement. Even with shallow sequencing, our loop velocity algorithm accurately gauges the trajectory of DNA motion and cellular states. The combined use of DropHiChew and loop velocity offers potential for a wide array of applications in future chromatin capture studies, including disease modeling, cellular differentiation studies, and developmental biology.

genomics↗

bioRxiv2024

Generation of High-fidelity Blastocyst-like Structures from Porcine Expanded Pluripotent stem cell via Chemically inducing cell plasticity

Understanding the mechanisms of blastocyst formation and implantation is crucial for advancing farm animal reproduction. However, research in this area is often hindered by the limited availability of embryos. In this study, we developed an efficient method to generate blastocyst-like structures (termed blastoids) from porcine expanded pluripotent stem cells (pEPSCs) through chemical induction with an efficiency up to 80%. These porcine blastoids closely resemble natural blastocysts in terms of morphology, cell composition, and single-cell transcriptomes. The tissue structures of the blastoids developed over time in culture, resembling the tissue morphology of gastrulation-stage embryos. This innovative approach not only provides a robust in vitro model for studying early embryogenesis in pigs but also holds the potential for improving reproductive efficiency and understanding the developmental processes of large farm animal species. The development of porcine blastoids represents a significant advancement in regenerative medicine and developmental biology.

cell biology↗

bioRxiv2024

Assessing the Relative Contributions of Mosaic and Regulatory Developmental Modes from Single-Cell Trajectories

Development is a highly complex process consisting of coordinated cell proliferation, cell differentiation and spatial organization. Classically, two ways to specify cell types during development are hypothesized : mosaic and regulative modes. In the mosaic mode, a particular cell isolated from the rest of the embryo will nevertheless give rise to cells with a fate identical to the ones expected in normal development, thus relying on lineage-inherited factors. In the regulative mode, the fate of a cell depends on its interactions with its environment, and thus relies on space-dependant factors. Both modes often coexist in the development of a given animal. We propose to quantify their respective contributions from single-cell trajectories. C. elegans development provides a unique opportunity to elaborate such an approach. Indeed, its invariant lineage enables the integration of spatial positions, lineage relationships and protein expression data. Using the single cell protein expression profile as a readout of the cell state, we relate the contributions of the mosaic and the regulative modes to the following measurable quantities. The contribution of the mosaic mode, or lineage-inherited contribution is quantified by the strength of the relationship between the cell-cell lineage distance and the cell-cell expression distance. Similarly, the contribution of the regulative mode, or context-dependent contribution is quantified by the strength of the relationship between the cell-cell context distance and the cell-cell expression distance. The cell-cell context distance measures the similarity between the spatial neighborhoods of two cells based on the gene expression profiles of their neighbours. We assess the significance of these contributions by comparing the empirical results obtained on C. elegans data to artificial models generated using simple rules. With these measures, we show the co-existence of mosaic and regulative modes in the development of C. elegans. The relative contribution of these two modes varies across the different tissues and in time. In particular, we see in the skin tissue that during early development, the mosaic mode dominates while at later stages, regulative mode dominates, suggesting a convergence of single cell trajectories. These measures are general and can be applied to other datasets that will be made available with the progress of spatial transcriptomics and lineage-tracing, paving the way for a quantitative, unbiased and perturbation-free study of fundamental concepts in developmental biology.

systems biology↗

bioRxiv2024

Get to know your neighbors with a SNAQ™: A framework for single cell spatial neighborhood analysis in immunohistochemical images

MotivationAnalyzing the local microenvironment of tumor cells can provide significant insights into their complex interactions with their cellular surroundings, including immune cells. By quantifying the prevalence and distances of certain immune cells in the vicinity of tumor cells through a neighborhood analysis, patterns may emerge that indicate specific associations between cell populations. Such analyses can reveal important aspects of tumor-immune dynamics, which may inform therapeutic strategies. This method enables an in-depth exploration of spatial interactions among different cell types, which is crucial for research in oncology, immunology, and developmental biology. ResultsWe introduce an R Markdown script called SNAQTM (Single-cell Spatial Neighborhood Analysis and Quantification), which conducts a neighborhood analysis on immunofluorescent images without the need for extensive coding knowledge. As a demonstration, SNAQTM was used to analyze images of pancreatic ductal adenocarcinoma. Samples stained for DAPI, PanCK, CD68, and PD-L1 were segmented and classified using QuPath. The resulting CSV files were exported into RStudio for further analysis and visualization using SNAQTM. Visualizations include plots revealing the cellular composition of neighborhoods around multiple cell types within a customizable radius. Additionally, the analysis includes measuring the distances between cells of certain types relative to others across multiple regions of interest. Availability and implementationThe R Markdown files that comprise the SNAQTM algorithm and the input data from this paper are freely available on the web at https://github.com/AryehSilver1/SNAQ. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/606539v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1cda240org.highwire.dtl.DTLVardef@157280forg.highwire.dtl.DTLVardef@1072cf8org.highwire.dtl.DTLVardef@1964e0_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com.

bioinformatics↗

bioRxiv2024

Optimizing Electroporation Parameters for Efficient Delivery of Large Molecules into Pig Zygotes Using Fluorescent Dextrans from 3 to 2000 kDA

Electroporation has revolutionized gene transfer and gene editing, enabling efficient delivery of molecules into embryos, with significant implications for developmental biology and biomedical research. This study aimed to optimize electroporation parameters for enhancing the delivery of large molecules into pig zygotes. We investigated the effects of fluorescence-coupled dextran reporters (FDs) of sizes ranging from 3 to 2000 kiloDalton (kDA) along with the impact of poring and transfer polarity settings during electroporation, on molecule permeability. Additionally, we assessed the influence of voltage and the number of poring pulses on the delivery of 2000 kDa FDs and examined the permeability of pre-IVF embryos and zona pellucida-weakened post-IVF embryos to this FD. Our findings highlighted size-dependent effects on FD uptake, with reversing poring polarity increasing the influx of small molecules (3 kDa FDs). The delivery of 2000 kDa FDs was not influenced by increased poring number but it was significantly influenced by voltage, reaching its optimum at 40 V. Electroporation in pre-IVF embryos did not show significant variation across different voltages. However, voltages higher than 20 V negatively affected blastocyst development rates. Zona-weakening did not improve permeability for the 2000 kDa FD. This study offers valuable insights into refining electroporation techniques for delivering large molecules into pig zygotes and highlights the relevance of commercial fluorescence-coupled dextrans as useful tools for exploring permeability dynamics in electroporated zygotes.

molecular biology↗

bioRxiv2024

Vascular network-inspired diffusible scaffolds for engineering functional neural organoids

Organoids, three-dimensional in vitro organ-like tissue cultures derived from stem cells, show promising potential for developmental biology, drug discovery, and regenerative medicine. However, the function and phenotype of current organoids, especially neural organoids, are still limited by insufficient diffusion of oxygen, nutrients, metabolites, signaling molecules, and drugs. Herein, we present Vascular network-Inspired Diffusible (VID) scaffolds to fully recapture the benefits of physiological diffusion physics for generating functional organoids and phenotyping their drug response. In a proof-of-concept application, the VID scaffolds, 3D-printed meshed tubular channel networks, support the successful generation of engineered human midbrain organoids almost without necrosis and hypoxia in commonly used well-plates. Compared to conventional organoids, these engineered organoids develop with more physiologically relevant features and functions including midbrain-specific identity, oxygen metabolism, neuronal maturation, and network activity. Moreover, these engineered organoids also better recapitulate pharmacological responses, such as neural activity changes to fentanyl exposure, compared to conventional organoids with significant diffusion limits. Combining these unique scaffolds and engineered organoids may provide insights for organoid development and therapeutic innovation.

bioengineering↗

bioRxiv2024

Effects of multistability, absorbing boundaries and growth on Turing pattern formation

Turing patterns are a fundamental concept in developmental biology, describing how homogeneous tissues develop into self-organized spatial patterns. However, the classical Turing mechanism, which relies on linear stability analysis, often fails to capture the complexities of real biological systems, such as multistability, non-linearities, growth, and boundary conditions. Here, we explore the impact of these factors on Turing pattern formation, contrasting linear stability analysis with numerical simulations based on a simple reaction-diffusion model, motivated by synthetic gene-regulatory pathways. We demonstrate how non-linearities introduce multistability, leading to unexpected pattern outcomes not predicted by the traditional Turing theory. The study also examines how growth and realistic boundary conditions influence pattern robustness, revealing that different growth regimes and boundary conditions can disrupt or stabilize pattern formation. Our findings are critical for understanding pattern formation in both natural and synthetic biological systems, providing insights into engineering robust patterns for applications in synthetic biology. Author summaryDuring development, tissues self-organize to go from a single cell to a structured organism. In this process, simple chemical reactions lead to the emergence of the intricate designs we see in nature, like the stripes on a zebra or the labyrinths on a brain cortex. Although multiple theories have been proposed to model this phenomenon, one of the most simple and popular ones was introduced in the 1950s by the mathematician Alan Turing. However, his theory oversimplifies the biological conditions and ignores properties such as non-linearities, boundary effects, or growth in the tissue. In this work, we used a combination of mathematical models and computer simulations to investigate how these real-world factors influence pattern formation. Our findings show that when we account for these realistic effects, the patterns that emerge can be very different from what Turings theory would predict. Thus, this work may help us better understand the laws behind pattern formation and could have practical applications in tissue engineering for medical or environmental applications.

biophysics↗

bioRxiv2024

Multi-color fluorescence live-cell imaging in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy.

cell biology↗