Biology subjects

Espinosa-Medina, I.

Publications and source records attributed to Espinosa-Medina, I..

10 recordsLinked to original sources

bioRxiv2026

Improved sensors for fructose-1,6-bisphosphate enable in vivo imaging of glycolysis

Fructose-1,6-bisphosphate (FBP) is the product of the first committed step of glycolysis, and its concentration is tightly correlated with glycolytic flux. Glycolytic activity varies across tissues and cell types: some tissues, such as the brain, dynamically regulate glycolysis in response to demand, while others, such as the liver have characterized spatial heterogeneity. Here, we report HYlight2, an improved sensor for FBP developed through random whole-gene mutagenesis in E. coli lysate. After four rounds of screening, we isolated HYlight2, which retains its binding affinity while displaying a {Delta}R/R [~]9 in vitro, a three-fold improvement in mammalian cells, and a two-fold improvement in detecting glycolytic responses during stimulated neuronal activity. We further demonstrate its use in vivo to detect altered glycolytic activity in C. elegans neurons, zebrafish pancreatic islets, and mouse liver.

biochemistry↗

bioRxiv2026

Reconstituting Mouse Embryogenesis Ex Utero from Gastrulation to Fetal Development Reveals Maternally Independent Metabolic Programs

Mammalian development takes place inside the maternal uterus, creating technological constraints that make difficult the study of embryogenesis in live developing embryos. A central challenge for understanding the role of metabolism in mammalian development is discriminating placental and uterine-regulated signals from embryo-intrinsic processes independent of maternal influence, a process that until now has remained inseparable during gastrulation and organogenesis1-3. Ex utero culture systems allowing continuous growth of embryos during pre-gastrulation to organogenesis4,5 offer a promising solution to this challenge. Here, we present optimized ex utero culture platforms that support faithful development of mouse embryos from gastrulation (embryonic day 6.5/7.5) through the fetal period (embryonic day [~]12.5) and harnessed these platforms for dissecting metabolic transitions in vivo during embryogenesis independently of uterus and placenta. We characterized the metabolome of in utero and ex utero whole embryos, fetal organs and culture medium between embryonic days E6.5 and E12.5 by liquid chromatography mass-spectrometry (LC-MS) metabolomics, isotope tracing, and single cell transcriptomics. These datasets present a comprehensive overview of the dynamic embryonic metabolism during gastrulation and organogenesis in utero and ex utero. This analysis revealed that the midgestational metabolic switch occurring at E10.5-E11.5 is faithfully recapitulated ex utero, indicating that this transition is intrinsically programmed in embryonic tissues and does not require direct maternal or placental cues. Notably, oxygen availability modulated the extent of this transition, but elevated oxygen was insufficient to induce it prematurely, demonstrating that the switch is developmentally timed and only partially environmental-responsive. We further harnessed the ex utero platform for identifying and perturbing a mitochondrial redox shift at E7.5-E8.5 that is critical for developmental progress after gastrulation. These findings uncover the remarkable metabolic plasticity of the mammalian embryo, demonstrating its capacity to sustain growth independently of maternal inputs from the establishment of the body plan through the onset of the fetal period. Moreover, they highlight the use of long-term ex utero culture as a unique framework for dissecting the mechanisms that shape embryogenesis under physiological and experimentally perturbed conditions, while functionally uncoupling embryonic programs from maternal and placental influences.

developmental biology↗

bioRxiv2026

mRNAbow: A versatile gene expression system for multiplexed fluorescent imaging using optimized in vitro transcribed mRNA

Messenger RNA (mRNA) transfection enables rapid, transient protein expression without nuclear entry, providing a powerful alternative to DNA or viral delivery in post-mitotic and otherwise difficult-to-transfect cells. Although in vitro transcribed (IVT) mRNAs have revolutionized therapeutic applications, their adoption in experimental biology remains limited by challenges in synthesis, variability across cell types, and concerns about cytotoxicity. Here, we define design principles that maximize IVT mRNA performance across diverse cellular and organismal systems. Through systematic comparison of capping strategies and base modifications, including N1-methyl-pseudouridine, 5-methylcytidine, and 5-methoxyuridine, we identify modifications that enhance translation while minimizing activation of cellular stress responses. Optimized transcripts drive robust protein expression within four hours, persist for up to one week, and support multiplexed expression of structurally and functionally distinct proteins in mammalian cells, including cancer cell lines, iPSC-derived systems, primary cells, and organoids, as well as in vivo in zebrafish embryos and in less genetically tractable models such as Danionella cerebrum and sea urchin embryos. To further expand accessibility for community use, we developed mRNAbow, a platform for generating low-toxicity mRNAs encoding organelle-targeted fluorescent proteins and biosensors for multiplex imaging, with corresponding plasmids made publicly available. Together, these advances establish a generalizable framework for IVT mRNA design and expand experimental access to synthetic mRNA technologies for dissecting cellular architecture and dynamics.

cell biology↗

bioRxiv2025

Imp1 acts as a dosage- and stage-dependent temporal rheostat orchestrating radial glial fate transitions and cortical morphogenesis

Cortical neurogenesis proceeds through a precise temporal program in which radial glia sequentially generate distinct neuronal subtypes and later glia, yet how post-transcriptional regulators coordinate these transitions remain poorly understood. We previously identified that a decreasing temporal gradient of the RNA-binding protein Imp encodes neural stem cell age in Drosophila. In this work, we extend our investigation to Imp1, a mammalian homologue of Imp, and its role in murine neocortical development. Using TEMPO to track birth-order dynamics, we demonstrate that sustained Imp1 overexpression during early neurogenesis arrests temporal fate progression, shifting neuronal populations toward deeper cortical layers V-VI. Immunostaining with layer-specific transcription factors Cux1 and Ctip2 confirmed that laminar repositioning results from genuine changes in neuronal identity rather than migratory defects, with neurons adopting molecular identities matching their final positions. Temporal window-specific manipulations reveal distinct stage-specific effects where early-stage Imp1 induction produces cascading effects on fate specification and moderately delays the neuronal-to-gliogenic transition, while mid-stage induction induces neuronal accumulation in the subplate region. Live imaging of organotypic cultures reveals continuous neuronal recruitment within intermediate and ventricular zones, with mid-stage-born neurons accumulating at significantly faster rates than earlier cohorts. Strikingly, mid-stage Imp1 overexpression also induces ectopic glial-like foci distributed throughout the cortical plate, featuring dramatic cellular expansion and morphological heterogeneity. These findings establish Imp1 as a dosage- and stage-dependent temporal rheostat orchestrating developmental transitions in radial glial progenitors, controlling neuronal fate decisions and spatial organization. This work advances our understanding of molecular timing mechanisms governing neuronal diversity in the mammalian cortex.

developmental biology↗

bioRxiv2024

Spatial single-cell Organellomics reveals nutrient dependent hepatocyteheterogeneity and predicts pathophysiological status in vivo

Cell state diversity drives tissue adaptability, repair, and disease resilience, but fully capturing this cellular complexity remains a challenge. Most current approaches rely on transcriptional profiling and often overlook functional insights embedded in organelle structure, key indicators of cellular metabolism and stress. Here, we introduce spatial Organellomics (sOrganellomics), an imaging workflow integrating automated segmentation with machine learning to classify and spatially map cell states based on multi-organelle features. Across metabolically specialized organs, these features distinguish organ-specific cellular identities. In the liver, sOrganellomics reveals a previously unrecognized hepatocyte organization: different hepatocyte states form intermixed communities rather than classical homogeneous graded zones, a pattern disrupted by nutritional stress. Intravital imaging further links fasting-associated architectural remodeling to altered mitochondrial membrane potential and bioenergetic heterogeneity in vivo. This approach maps metabolic transitions and early disease progression, establishing organelle architecture as a scalable readout of functional cell state diversity in tissues.

cell biology↗

bioRxiv2021

TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification. One-Sentence SummaryGaining sequential genetic access to vertebrate cell lineages.

developmental biology↗

bioRxiv2020

Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling

Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.

developmental biology↗

bioRxiv2019

Transcriptional reprogramming in fused cells is triggered by plasma-membrane diminution

Developing cells divide and differentiate, and in many tissues, such as bone, muscle, and placenta, cells fuse acquiring specialized functions. While it is known that fused-cells are differentiated, it is unclear what mechanisms trigger the programmatic-change, and whether cell-fusion alone drives differentiation. To address this, we employed a fusogen-mediated cell-fusion system involving undifferentiated cells in tissue culture. RNA-seq analysis revealed cell-fusion initiates a dramatic transcriptional change towards differentiation. Dissecting the mechanisms causing this reprogramming, we observed that after cell-fusion plasma-membrane surface area decreases through increased endocytosis. Consequently, glucose-transporters are internalized, and cytoplasmic-glucose and ATP transiently decrease. This low-energetic state activates AMPK, which inhibits YAP1, causing cell-cycle arrest. Impairing either endocytosis or AMPK prevents YAP1 inhibition and cell-cycle arrest after fusion. Together these data suggest that cell-fusion-induced differentiation does not need to rely on extrinsic-cues; rather the plasma-membrane diminishment forced by the geometric-transformations of cell-fusion cause transient cell-starvation that induces differentiation.

cell biology↗

bioRxiv2019

CLADES: a programmable sequence of reporters for lineage analysis

We present CLADES (Cell Lineage Access Driven by an Edition Sequence), a technology for cell lineage studies based on CRISPR/Cas9. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a pre-determined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, coupling birth order with reporter expression. This gives us temporal resolution of lineage development that can be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, marking each generation with the corresponding combination of reporters. CLADES thus offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.\n\nOne Sentence SummaryA sequence of reporters for lineage analysis

developmental biology↗

bioRxiv2018

Unlimited genetic switches for cell-type specific manipulation

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions, as well as to deliver precise gene therapy. Transcriptome analyses have allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types have remained inaccessible to genetic manipulation. Here, we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches that each respond to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell type-specific manipulations and combinatorial genetic targeting. CaSSA is thus a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

developmental biology↗