Biology subjects

Mau, C.

Publications and source records attributed to Mau, C..

5 recordsLinked to original sources

bioRxiv2026

Single-cell transcriptomics of enriched rare neural lineages of Tribolium castaneum

Background Single-cell transcriptomic atlases have transformed our understanding of cell diversity. However, studies designed to profile entire tissues or organisms often omit or underrepresent rare cell populations. Enriching selected populations before sequencing offers a powerful approach to overcoming this limitation, but its application in insects has largely focused on Drosophila, limiting opportunities for comparative studies. Here, we establish complementary workflows for targeted transcriptomic profiling of rare neural populations in the red flour beetle, Tribolium castaneum. Results We developed two workflows combining population enrichment with single-cell or single-nucleus RNA sequencing. First, we generated cell suspensions from a transgenic reporter line labelling embryonic neuroblasts, enriched fluorescently labelled cells by fluorescence-activated cell sorting, and performed single-cell RNA sequencing. Second, we immunolabelled first-instar larval brains with an antibody against the nuclear transcription factor Rx, isolated and sorted labelled nuclei, and performed single-nucleus RNA sequencing. Both workflows yielded material suitable for transcriptomic profiling, demonstrating complementary reporter-based and antibody-based strategies for accessing selected neural populations at different developmental stages. Conclusions These workflows establish a methodological framework for targeted single-cell and single-nucleus transcriptomic studies of rare or specific neural populations in Tribolium. They provide practical starting points for adaptation to other insect systems, expanding opportunities for comparative investigations of neural development and cell-type evolution.

developmental biology↗

bioRxiv2026

Temporal-to-spatial patterning of embryonic structures can involve active transformation of temporal information rather than direct mapping

How spatial patterns arise during embryonic development is classically explained by the French Flag model, in which cells acquire positional identities by interpreting morphogen concentration thresholds. However, in many developmental systems, spatial patterns instead emerge progressively through temporal programs of gene expression that are transformed into spatial organization. For example, in the short-germ insect Tribolium castaneum, both periodic pair-rule gene expressions that generate body segments and non-periodic gap gene expressions that establish regional identities arise sequentially at the posterior and propagate anteriorly in waves across the developing embryo. Understanding how such temporal gene expression programs are translated into spatial patterns remains a major challenge. To address this problem, we developed a sequential multiplexed imaging strategy based on hybridization chain reaction (HCR), enabling visualization of up to ten anterior-posterior (AP) patterning genes within the same Tribolium embryo. By combining this approach with intronic-exonic labeling, we established a framework to infer gene expression dynamics and propagatory behavior during AP patterning. Using this framework, we show that gap gene expression domains remain dynamic and continue to propagate during tissue elongation, indicating that spatial patterns are actively remodeled throughout development. We then directly compared temporal gene activation at the posterior with the resulting spatial organization of pair-rule and gap genes. Surprisingly, while primary pair-rule genes preserve their temporal phase relationships in space, gap genes do not. Instead, the relative positioning of gap gene domains progressively changes as they move anteriorly, indicating that the final spatial organization of gap genes is actively reshaped during propagation rather than being directly inherited from the initial temporal sequence. The continued propagatory behavior of gap gene domains suggests that such reshaping could arise through differential propagation dynamics between genes and/or through progressive reconfiguration of underlying gene regulatory interactions during pattern formation. Together, these findings reveal that temporal-to-spatial patterning can involve active transformation of temporal information rather than a simple mapping from time into space.

developmental biology↗

bioRxiv2026

Enhancer RNA Transcription Near Segmentation Gene Enhancers Can Be Analyzed In Situ Using FISH

Enhancer RNAs (eRNAs) are non-coding transcripts produced at enhancer regions, which appear to be involved in transcriptional regulation. Up to date, these have been primarily investigated using labor-and cost-intensive genomic techniques. However, the precise mechanisms by which eRNA transcription or the eRNA transcripts themselves mediate transcriptional regulation remain unclear. Here, we present a novel experimental approach that allows us to analyze the characteristics of eRNA transcription in fixed and live whole Drosophila melanogaster embryos. We employ the anterior-posterior patterning genes as a model system to investigate the dynamics of eRNA expression, utilizing an imaging-based approach. We combined high-sensitivity fluorescence in situ hybridization (FISH) chain reaction (HCR) with high-resolution confocal microscopy to detect eRNA and mRNA molecules. Through this experimental assay, we identified foci of elevated transcriptional activity that generate eRNA transcripts correlated with mRNA production at the same gene locus. We could show that this eRNA transcription is independent of promoter activity. Additionally, we demonstrate that insulators can influence eRNA transcription, resulting in loss of eRNA transcription. Moreover, we observe that eRNAs can originate both within classical enhancer regions and outside of them, including from foreign bacterial sequences when these are placed near enhancer sequences, underscoring the strong influence of local regulatory context on eRNA initiation. In live embryos using MS2-MCP live imaging, our analysis of insulators showed a modest reduction in mRNA burst intensity accompanied by a slight increase in burst frequency. Overall, our imaging-based approach offers a novel platform for dissecting enhancer-eRNA interactions and could be adapted for wider applications.

developmental biology↗

bioRxiv2025

Gene network switching provides a mechanistic basis for time-to-space translation in insect embryonic patterning

The French Flag model has long served as the prevailing framework for explaining how morphogen gradients generate spatial domains during embryonic development. More recently, however, evidence has shown that many tissues instead establish patterns by translating the sequential activation of genes (organized into genetic cascades) into spatial domains. This translation is thought to occur through modulation of the speed or timing of cascade progression, but the mechanisms underlying such temporal control remain unclear. Two models have been proposed: the general kinetic modulation model, in which morphogens influence global kinetic factors such as transcription and decay rates, and the gene regulatory network (GRN) switching model, in which morphogens reconfigure regulatory interactions so that genes initially function within a genetic cascade driving sequential activation, but are later integrated into a stabilizing network that locks their expression into mutually reinforcing domains. This transition is hypothesized to occur through a shift from a dynamic GRN (a genetic cascade that drives sequential activations) to a static GRN (a stabilizing network that maintains gene expression domains). Using gap genes in Tribolium castaneum as a model, we combined HCR in situ hybridization, parental RNA interference, and computational modeling to test these hypotheses. We show that gap genes initially act in a genetic cascade producing sequential activations, followed by a morphogen-dependent reconfiguration that stabilizes spatial domains. In particular, we identify the Mlpt-Svb complex as a key stabilizing factor that maintains svb expression anteriorly after its initial activation in the posterior. Computational simulations reproduce experimental phenotypes and support GRN switching as the underlying mechanism. Together, these findings demonstrate how morphogen-driven rewiring of network interactions converts temporal cascades into stable spatial patterns, providing a mechanistic basis for robust anterior- posterior patterning in insect embryos and beyond.

developmental biology↗

bioRxiv2022

How enhancers regulate wavelike gene expression patterns: A novel enhancer prediction and live reporter system identifies an enhancer associated with the arrest of pair-rule waves in Tribolium

A key problem in development is to understand how genes turn on or off at the right place and right time during embryogenesis. Such decisions are made by non-coding sequences called enhancers. Much of our models of how enhancers work rely on the assumption that genes are activated de novo as stable domains across embryonic tissues. Such view has been strengthened by the intensive landmark studies of the early patterning of the anterior-posterior (AP) axis of the Drosophila embryo, where indeed gene expression domains seem to arise more or less stably. However, careful analysis of gene expressions in other model systems (including the AP patterning in vertebrates and short-germ insects like the beetle Tribolium castaneum) painted a different, very dynamic view of gene regulation, where genes are oftentimes expressed in a wavelike fashion. How such gene expression waves are mediated at the enhancer level is so far unclear. Here we establish the AP patterning of the short-germ beetle Tribolium as a model system to study dynamic and temporal pattern formation at the enhancer level. To that end, we established an enhancer prediction system in Tribolium based on time- and tissue-specific ATAC-seq and an enhancer live reporter system based on MS2 tagging. Using this experimental framework, we discovered several Tribolium enhancers, and assessed the spatiotemporal activities of some of them in live embryos. We found our data consistent with a model in which the timing of gene expression during embryonic pattern formation is mediated by a balancing act between enhancers that induce rapid changes in gene expressions (that we call dynamic enhancers) and enhancers that stabilizes gene expressions (that we call static enhancers).

developmental biology↗