The mission of our lab

In the Giraldez Lab, we combine genetics, embryology, genomics, biochemistry, computational biology, and RNA engineering to address a central question in biology: how does a fertilized egg develop into a complex multicellular embryo?

Our research focuses on the maternal-to-zygotic transition (MZT), a fundamental process in developmental biology that occurs in all animals. This transition involves two main steps: first, during the maternal stages, the zygotic genome is transcriptionally silent, and early development is controlled by maternally deposited mRNAs and proteins. Then, the activation of the zygotic genome occurs, leading to the clearance of these maternal mRNAs and the progression to zygotic development.

We aim to answer key questions such as: How is the zygotic genome activated? What triggers the decay of maternal mRNAs during the transition to zygotic stages? How do microRNAs (miRNAs) and other non-coding RNAs regulate gene expression during development? And importantly, how can we leverage this understanding of post-transcriptional regulation to improve mRNA therapies and engineer better therapeutic mRNAs with tissue specificity?

Our lab also applies this knowledge to the development of therapeutic mRNAs. Using cutting-edge genomics, massively parallel reporter assays, and computational approaches, we engineer mRNAs for gene therapy and vaccine development. This research aims to create more precise and effective therapeutic mRNAs with applications in treating genetic disorders and enhancing immune responses.?

Our research

We aim to define the regulatory code that shapes embryogenesis, and use this knowledge to develop therapeutic mRNAs for gene therapy and vaccine development.


Zygotic genome activation

 CRISPR/Cas9 is adapted to label genomic loci (here, mirR-430 is labelled on each chromosome) over the course of cell divisions in living zebrafish embryos. Each frame is presented in maximum projections.

In all animals, maternal mRNAs and proteins deposited in the egg direct the initial stages of development and the embryos genome is transcriptionally silent. This set of “instructions” - called the maternal contribution - is fundamental to the development of every organism.

The transition from maternal-to-zygotic nuclear control represents a key event in developmental reprogramming. This process requires chromatin remodeling to establish transcriptional competency, maternal transcription factors to specifically activate the new transcriptional program and the clearance and degradation of maternal products. We are interested in understanding how these molecular events license the genome for activation using the genetic tools available to the zebrafish system in combination with biochemical and high throughput genomic approaches.

We have recently identified three key maternal transcription factors – Nanog, SoxB1 (Sox2) and Pou5f3 (Oct4) – as being widespread regulators of gene activation during this transition in zebrafish (Lee, Bonneau et al Nature 2013). Loss of these factors results in complete developmental arrest and failure to activate ~ 80% of zygotic genes. We are now interested in understanding the molecular mechanism by which these factors direct activation and mediate genome competence during this fundamental transition in biology.

Post-transcriptional Regulation

 During the Maternal-to-Zygotic Transition, a combination of microRNAs, RNA Binding Proteins, and other post-transcriptional regulators drive decay of maternal mRNA and zygotic genome activation.

Post-transcriptional control of gene expression is a fundamental mechanism of cellular function involved in all aspects development, and maintenance of the healthy state in eukaryotic organisms. The earliest stages of animal development occur in the absence of transcription, which offers a unique context to study specifically post-transcriptional mechanisms of gene regulation. Using the zebrafish Danio rerio and the African claw-toed frog Xenopus as a model organisms, we use functional genomics to understand the post-transcriptional regulatory code in vertebrates. Our efforts span investigating RNA stability, RNA modifications, RNA structure, RNA binding proteins and their recognition sequences, upstream ORFs, non-coding RNAs, and translation regulation. We aim to understand the role for these regulatory features during embryonic development to gain insight into how a single cell gives rise to a multicellular organism.

Our starting point to investigate the post-transcriptional regulatory code is the maternal to zygotic transition, a universal developmental transition across animal development where thousands of mRNAs are post-transcriptionally regulated. While microRNAs play a role in this regulation, these can only explain ~20% of observed mRNA dynamics (Giraldez, A et al. Science. 2006.). Our goal is to identify the factors responsible for the remaining 80%. We are currently investigating how translation by the ribosome, RNA binding proteins and non-coding RNAs regulate mRNAs for decay.

Furthermore we have developed methods to probe the structure of the RNA, the function of individual RNA fragments in the transcriptome, and the proteins bound to the RNA. Combining iCLIP with motif discovery algorithms we are defining the regulatory network of proteins that recognize specific sequences and structures to regulate mRNA stability and translation. We have also recently found an important role for the ribosome in the regulation of mRNA stability and translational regulation (Bazzini, A et al. EMBO J. 2016).

Using this information we are using machine learning algorithms to integrate each of the regulatory inputs mentioned above to model gene expression during developmental transitions across species.

Finally, we aim to understand the role for each of those elements in embryogenesis. We are capitalizing on recent improvements in CRISPR technologies (Moreno-Mateos MA⋆ and Vejnar CE⋆, et al., Nature Methods. 2015.) to mutagenize these elements and understand their function in development.


Computational Modeling of Gene Regulation

 We developed an assay to identify regulatory regions in the Zebrafish genome. We are now developing models to predict the activity of these regions during development. (Yartseva et al. Nature Methods. 2017.)

Post-transcriptional regulation is critically important in determining cellular phenotypes and behavior, particularly during early development when the genome is transcriptionally silent. One focus in our lab is to combine novel high-throughput techniques to dissect regulatory elements in the genome, with computational analysis and modeling to dissect the various regulatory programs that control vertebrate gene expression.

Our most recent work in this area has identified micropeptides and upstream open reading frames, which are widespread throughout the vertebrate transcriptome. Using ribosome profiling, RNA-seq, and reporter assays, we showed that uORFs are a prevalent regulatory mechanism by which the cell represses the translation of thousands of proteins. We were also able to computationally identify the sequence features most predictive of repression, and show that the activity of uORFs is conserved across species.

Our ultimate goal is to combine our knowledge of the various regulatory mechanisms in the early embryo, such as miRNAs, uORFs, codon optimality, RNA structure, and the RBP interactome, to form a comprehensive and predictive model of translation regulation.

Development of Therapeutic mRNAs

In our lab, we are leveraging insights into RNA biology to engineer therapeutic mRNAs with enhanced stability, tissue specificity, and controlled expression. By combining high-throughput techniques, such as massively parallel reporter assays and RNA-seq, with computational modeling, we aim to identify and optimize the sequence and structural elements that govern mRNA stability and translation efficiency. One of our key focuses is to understand how these regulatory elements, including miRNAs, RNA-binding proteins (RBPs), and codon usage, can be harnessed to precisely control mRNA behavior in vivo. Using lipid nanoparticles (LNPs) as delivery vehicles, we are developing therapeutic mRNAs for gene therapy and vaccine applications. Our computational models are critical in predicting the functionality of these mRNAs in different tissues, allowing us to design therapeutic molecules with improved tissue specificity. Ultimately, our goal is to apply this comprehensive understanding of mRNA regulation to create more effective and targeted RNA-based therapies for a range of diseases.