In the Giraldez lab we combine genetics, embryology, genomics, biochemistry, and computational biology to address a central question in biology: how does a fertilized egg develop into a complex multicellular embryo?
In particular, we are interested in a long standing problem in developmental biology the maternal to zygotic transition (MZT). This universal transition takes place in all animals and consists of two main steps. First, the maternal stages are characterized by a transcriptionally silent zygotic genome, where the first developmental decisions depend on the maternally deposited mRNAs and proteins. Next, activation of the zygotic genome takes place and this triggers the clearance of maternally deposited mRNAs to progress to zygotic stages.
We aim to understand the following questions: How is the zygotic genome activated? What are the factors that trigger the decay of maternal mRNAs to undergo zygotic development? How do miRNAs and other non-coding RNAs regulate gene expression during development?
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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.
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 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.
Autism is a genetic neurodevelopmental disease. In the Giraldez Lab we investigate the genes linked to Autism with the goal of identifying the molecular, cellular and electrophysiological defects caused by these mutations in zebrafish embryos. While the analysis of Autism and other neurodevelopmental human syndromes might sound far fetched, there are tremendous advantages of using a vertebrate model system with rapid development and complex behavioral phenotypes, whose behaviour can be monitored in large numbers. More importantly we have adapted behavioural profiling with chemical screening to characterize the phenotypes of mutants and identify pharmacological compounds that might suppress the behavioural phenotypes and inform us of the pathways affected in these mutants with the ultimate goal of identifying supressor drugs for this syndrome.
In a recent study (Hoffman et al., Neuron 2016) we have investigated the function of Cntnap2, a gene linked to Autism. Using high-throughput behavioral profiling we identified nighttime hyperactivity in cntnap2 mutants, while pharmacological and neurodevelopmental assays reveal dysregulation of GABAergic and glutamatergic systems. Combining behavioral profiling with a pharmacological screening identified estrogenic compounds as phenotypic suppressors, suggesting that these compounds serve as modifiers of neural circuits disrupted in mutants. This study provides an entry point to characterize additional mutations associated with autism and define common suppressors as potential therapeutic agents for further investigation.