About

In the Giraldez Lab, we study the regulatory code that governs gene expression during vertebrate development after fertilization. We focus on genome activation, post-transcriptional regulation, modeling of therapeutic mRNA design, and modeling of gene regulatory networks. Our research combines super resolution imaging, genomics, and computational methods to understand gene regulation and its applications in gene therapy and vaccine development.

Research topics

• Genetics
• Biology
• Cell biology
• Computer Science
• Computational biology
• Artificial Intelligence
• Physics
• Algorithm
• Biological system
• Chemistry
• Mathematics
• Molecular biology

Selected publications

• Lamin A/C directs nucleosome-scale chromatin remodeling to define early lineage segregation in mammals

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-01-02

  articleOpen accessSenior authorCorresponding

  Chromatin organization underlies gene regulation and cell fate specification, yet how nucleosome-scale chromatin structure contributes to lineage segregation during early development remains unknown. Here, we resolved chromatin ultrastructure during the first lineage decision in mouse and human, which forms the pluripotent inner cell mass (ICM) and trophectoderm (TE). To achieve this, we developed dual-tilt chromatin electron tomography (2T-ChromEMT) that allows multiscale visualization of chromatin architecture. Our analysis reveals that TE cells of both species display denser chromatin with nucleosome aggregation at the nuclear periphery. We show upregulation of the nuclear matrix protein Lamin A/C within the TE lineage across mouse, human, and opossum embryos, indicating that its regulatory role is conserved across eutherian and marsupial species. Loss of Lamin A/C reduces heterochromatin at the nuclear lamina in TE cells, reactivates pluripotency genes, and impairs mouse blastocyst expansion and human blastoid formation. These findings define the nucleosome-resolution chromatin signatures of early mammalian lineages and establish Lamin A/C-mediated chromatin organization as a conserved mechanism in the exit from pluripotency and maintenance of trophectoderm identity.

  PublisherOA PDFDOI

• RNAGenScape: Property-Guided, Optimized Generation of mRNA Sequences with Manifold Langevin Dynamics.

  PubMed · 2026-05-08

  article

  Generating property-optimized mRNA sequences is central to applications such as vaccine design and protein replacement therapy, but remains challenging due to limited data, complex sequence-function relationships, and the narrow space of biologically viable sequences. Generative methods that drift away from the data manifold can yield sequences that fail to fold, translate poorly, or are otherwise nonfunctional. We present RNAGenScape, a property-guided manifold Langevin dynamics framework for mRNA sequence generation that operates directly on a learned manifold of real data. By performing iterative local optimization constrained to this manifold, RNAGenScape preserves biological viability, accesses reliable guidance, and avoids excursions into nonfunctional regions of the ambient sequence space. The framework integrates three components: (1) an autoencoder jointly trained with a property predictor to learn a property-organized latent manifold, (2) a denoising autoencoder that projects updates back onto the manifold, and (3) a property-guided Langevin dynamics procedure that performs optimization along the manifold. Across three real-world mRNA datasets spanning two orders of magnitude in size, RNAGenScape increases median property gain by up to 148% and success rate by up to 30% while ensuring biological viability of generated sequences, and achieves competitive inference efficiency relative to existing generative approaches.

  Publisher

• Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo

  Nature Communications · 2025-07-01 · 5 citations

  articleOpen accessSenior author

  Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community. Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, using a toolkit consisting of genetically-encoded epitope probes, their cognate tags, and an array of adapter proteins, the authors describe a methodology that enables visualization and manipulation of endogenous proteins in vertebrate systems.

  PublisherOA PDFDOI

• G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

  Cell Reports · 2025-02-01 · 8 citations

  articleOpen access

  The subcellular localization of mRNAs plays a pivotal role in biological processes, including cell migration. For instance, β-actin mRNA and its associated RNA-binding protein (RBP), ZBP1/IGF2BP1, are recruited to focal adhesions (FAs) to support localized β-actin synthesis, crucial for cell migration. However, whether other mRNAs and RBPs also localize at FAs remains unclear. Here, we identify hundreds of mRNAs that are enriched at FAs (FA-mRNAs). FA-mRNAs share characteristics with stress granule (SG) mRNAs and are found in ribonucleoprotein (RNP) complexes with the SG RBP. Mechanistically, G3BP1 binds to FA proteins in an RNA-dependent manner, and its RNA-binding and dimerization domains, essential for G3BP1 to form RNPs in SG, are required for FA localization and cell migration. We find that G3BP1 RNPs promote cell speed by enhancing FA protein mobility and FA size. These findings suggest a previously unappreciated role for G3BP1 RNPs in regulating FA function under non-stress conditions.

  PublisherDOI

• A Multi-Modal and Generalizable Nanogold Tag for Cryo-Electron Tomography in Intact Cells

  Structural Dynamics · 2025-03-01

  articleOpen access

  Cryo-Focused Ion Beam-Electron Tomography (cryo-FIB-ET) is quickly becoming a powerful tool for investigating structure- function relationships in native environments, enabling the discovery of new biology. However, this technique is often limited to visual identification of unambiguously large macromolecules (whole organelles, ribosomes, the nuclear pore complex, proteasomes), or through the matching of high resolution signatures (2D template matching) of sufficient molecular weight. The need for additional tagging methods is paramount, in order to identify macromolecules and expand our scientific inquiries. We have developed a delivery and tagging system utilizing gold nanoparticles that specifically targets macromolecules to be visualized by cryo-FIB-ET and resolved on a per-particle basis by subtomogram analysis. This method has been optimized on a ribosomal target as a proof-of-principle, and then further applied to in situ heterochromatin by tagging specific histone variants associated with heterochromatin. The labeling, delivery, and identification strategy presented here would facilitate future progress for identifying smaller macromolecules and distinguishing between highly homologous molecules, with minimal perturbations and imaged within the cell at high spatial resolution.

  PublisherDOI

• In vivo differentiation of embryonic cells devoid of key reprogramming factors

  Cell Reports · 2025-10-30 · 1 citations

  articleOpen access

  Embryonic cell differentiation depends on reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this coincides with genome activation, which is regulated by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Here, we investigate the role of NPS in developmental reprogramming and differentiation by analyzing the fate of NPS mutant cells in a wild-type embryo using single-cell RNA-seq. We find that many cells fail to activate transcription or undergo cell death, while others acquire gene expression profiles that resemble germ cells, neural progenitors, and motoneuron states. These cells achieve intermediate transcriptional states, revealing the essential role of NPS in coordinating nuclear and cytoplasmic reprogramming and preventing the premature activation of lineage-specific differentiation programs. These results demonstrate that most developmental programs require developmental reprogramming by NPS, yet some cells can bypass transient totipotency to achieve intermediate developmental states resembling wild-type states in vivo.

  PublisherOA PDFDOI

• ExoSloNano: multimodal nanogold labels for identification of macromolecules in live cells and cryo-electron tomograms

  Nature Methods · 2025-11-28 · 1 citations

  articleOpen access

  In situ cryo-electron microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Recent progress in sample preparation, imaging and data processing has enabled the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify macromolecules across multiple size scales (from microns to nanometers). Here we introduce nanogold probes for detecting specific proteins using correlative light and electron microscopy, cryo-electron tomography (cryo-ET) and resin-embedded electron microscopy. These nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this ExoSloNano system to identify both cytoplasmic and nuclear proteins by room-temperature electron microscopy, and resolve associated structures by cryo-ET. By providing high-efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, ExoSloNano expands the proteome available to electron microscopy.

  PublisherDOI

• RNADecayCafe, a uniformly processed atlas of RNA half-life estimates across multiple human cell lines

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-21 · 2 citations

  preprintOpen access

  RNA expression levels are partially determined by RNA stabilities. Long-lived RNAs accumulate to higher levels than rapidly degraded RNAs when transcribed at the same rate. The extent to which RNA decay contributes to differences in gene expression between genes in the same cell or between different cell types has not been extensively examined. This is in part because reproducible RNA half-life estimates from different biological contexts have not been broadly collected and curated. To address this, we have developed RNAdecayCafe, a database of high-quality RNA half-life estimates in multiple human cell lines. RNAdecayCafe makes use of pulse-label nucleotide recoding RNA-seq data (namely SLAM-seq and TimeLapse-seq), which we found to provide consistent and orthogonally validated half-life estimates. In total, RNAdecayCafe provides half-life estimates from 11 different human cell lines across 15 datasets and a total of 64 samples that were uniformly processed with a set of optimized bioinformatic tools we recently developed (the EZbakR suite). We showcase how this resource reveals the previously underappreciated role of RNA stability in shaping gene expression. RNAdecayCafe will provide a robust database for future studies of RNA decay.

  PublisherOA PDFDOI

• Maternal regulation of the vertebrate oocyte-to-embryo transition

  PLoS Genetics · 2024-07-25 · 5 citations

  articleOpen accessCorresponding

  Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.

  PublisherOA PDFDOI

• UPF1 regulates mRNA stability by sensing poorly translated coding sequences

  Cell Reports · 2024-04-01 · 16 citations

  articleOpen accessSenior authorCorresponding

  Post-transcriptional mRNA regulation shapes gene expression, yet how cis-elements and mRNA translation interface to regulate mRNA stability is poorly understood. We find that the strength of translation initiation, upstream open reading frame (uORF) content, codon optimality, AU-rich elements, microRNA binding sites, and open reading frame (ORF) length function combinatorially to regulate mRNA stability. Machine-learning analysis identifies ORF length as the most important conserved feature regulating mRNA decay. We find that Upf1 binds poorly translated and untranslated ORFs, which are associated with a higher decay rate, including mRNAs with uORFs and those with exposed ORFs after stop codons. Our study emphasizes Upf1's converging role in surveilling mRNAs with exposed ORFs that are poorly translated, such as mRNAs with long ORFs, ORF-like 3' UTRs, and mRNAs containing uORFs. We propose that Upf1 regulation of poorly/untranslated ORFs provides a unifying mechanism of surveillance in regulating mRNA stability and homeostasis in an exon-junction complex (EJC)-independent nonsense-mediated decay (NMD) pathway that we term ORF-mediated decay (OMD).

  PublisherOA PDFDOI

Recent grants

• Functional analysis of the zebrafish genome through RNA-seq and ribosome profile

  NIH · $1.8M · 2012–2018

• NIH Grant R01GM102251

  NIH · $1.3M · 2017

• NIH Grant R21HD073768

  NIH · $445k · 2015

• NIH Grant R01GM103789

  NIH · $1.3M · 2017

• NIH Grant R01GM101108

  NIH · $1.5M · 2017

Frequent coauthors

• Alexander F. Schier

  University of Basel

  91 shared

• Anton J. Enright

  University of Cambridge

  54 shared

• Charles E. Vejnar

  Yale University

  52 shared

• Margaret E. Glasner

  Texas A&M University

  36 shared

• Scott Baskerville

  36 shared

• Scott M. Hammond

  University of North Carolina at Chapel Hill

  36 shared

• David P. Bartel

  Whitehead Institute for Biomedical Research

  36 shared

• Ryan M. Cinalli

  Icahn School of Medicine at Mount Sinai

  36 shared

Labs

• Giraldez LabPI

  Investigates the regulatory codes that shape gene expression during embryonic development after fertilization.