Research Projects

Activation of the zygotic genome:

       In all animals, the mother deposits mRNAs and proteins in the egg so that the embryo can undergo development until the activation of its own genome occurs. This set of “instructions” -called the maternal contribution- and is fundamental to the development of every organism. But what triggers the activation of the zygotic genome? To answer this question we have first identified the first set of zygotically expressed genes. Because a large fraction of the genome is maternally expressed, this is not a trivial task. We have used the intronic signal to define the first set of zygotically expressed mRNAs that depend on the maternal products. Based on the translation profile of all sequence specific transcription factors, we have identified nanog, oct4 and soxB1 as the most highly translated in the early embryo before MZT. Indeed, Chromatin IP and loss of function experiments reveal that the first zygotic genes are strongly enriched for binding of these factors and they are requeired for expression of a large fraction of the first zygotic genes. We are now investigating how these factors mediate genome competence during this fundamental transition in biology.

        In parallel we are developing novel genetic screens to to eliminate the maternal contribution and identify the genes first required for zygotic development.

Regulation of the maternal mRNAs during the maternal-to-zygotic transition:

      Upon activation of the zygotic program, these maternal instructions are degraded, but the mechanism that selects some mRNAs for degradation has remained elusive. In 2006, we identified for the first time that miRNAs play an important role in this process, selecting a large fraction of the maternal mRNAs for repression and degradation. In particular, miR-430 has the potential to regulate up to 40% of the maternal mRNAs in zebrafish. In this project we are also searching the maternal mRNAs that lack miR-430 sites to identify  novel sequences that regulate maternal genes by combining genomics and proteomics. We call this pathway Zyfir, for zygotic factors that mediate repression and decay, and we believe that the factors and regulatory elements that mediate this interaction are likely to play central roles not only in development but also in every context where RNA regulation in required. 

Deciphering the post-transcriptional regulatory code:

We have developed novel high throughput methods to interrogate the regulatory potential of individual elements in the transcriptiome, including sequence and structural elements. Indeed we are currently combining computational and  high throughput sequencing approaches to characterize the RNA structure of the transcriptome, to define a common regulatory code.  Finally we are Using iCLIP and Biochemical purification to identify the RNA-protein interaction network that reads the regulatory code to shape post-transcriptional regulation during verterbate development.


The role of non-coding RNAs in Vertebrate Development


        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.

        In the Giraldez’ lab we aim to understand: How is the zygotic genome activated? what are the factors that trigger the decay of maternal mRNAs to undergo zygotic development? and how do miRNAs and other non-coding RNAs regulate gene expression during development?


Crispr nucleases

We are engineeting a variety of tools to generate loss-of-function mutants in several zebrafish genes of interest, including non-coding RNAs, micropeptide encoding genes, RNA binding factors, and regulatory elements in the RNA.

We have also developed computational methods to identify the most effieicnt sgRNA/Cas9 complexes for gene targeting in vivo. CRISPRscan

Maternal Zygotic mutants

Using the germ line replacement technique, we can generate wild type adult fish where the germ line is homozygous mutant for dicer or other maternally provided genes. This allows us to eliminate the maternal contribution of these genes.

High throughput sequencing

We are using high throughput sequencing, ribosome profiling and iCLIP to identify novel non-coding RNAs, characterize gene transcription and translation across the genome with the ultimate goal of defining the functional elements during development.

“how does an embryo develop from a fertilized egg”

miRNA ~30%

Y          >60%



Zygotic stages:

activation of transcription

maternal mRNAs are degraded

Maternal stages:

transcriptionally silent

maternal mRNAs

Top: central dogma of molecular biology adapted to the maternal-to-zygotic transition. Currently we do not know what factors clear the maternal mRNAs (Y) and activate the zygotic transition (X)

Example of an mRNA that is cleared during the maternal to zygotic transition based on the activity of miR-430. (Carter Takacs)

Characterization of translational regulation during embryonic development:

We have used ribosome profiling (a technique developed by Nick Ingolia and Jonathan Weissman) to characterize the dynamics of translational regulation during embryonic development. This is allowing us to define factors that regulate gene expression post-transcriptionally as well as defining the coding potential of the genome. Indeed, we have identified micropeptide encoding genes and regulatory upstream open reading frames that we are currently studying to aunderstand their function in development and gene regulation.

Nanog + Oct4



RNA Binding Proteins

       Our most recent work in this area has identified micropeptides (upper panel) and upstream open reading frames (uORFs, lower panel), 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.

The goal of our research is to identify the function of these coding sequences in development by generating targeted mutants using CRISPR/Cas9 nucleases. Furthermore, we are using the regulatory information in vivo to develop computationals models of gene expression.

Figure: Combining high resolution ribosome footprinting with computational methods to detect translation has allowed us to identify of translated regions across predicted non-coding regions, including micropeptides, (small ORFs, below) and upstream ORFs (below), as well as quantification of the translation for individual mRNAs across development