Research Projects
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Clearance of the maternal mRNAs during the maternal-to-zygotic transition:

        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. 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. Interestingly, miR-430 has the same seed sequence than other vertebrates miRNAs including, miR-302, miR-372, miR-295, miR-17 and miR-428. These miRNAs are all expressed in early development in vertebrates and ES cells and are important in cellular reprograming. miR-17 and miR-372 can cause cancer in humans. We are studying how the regulation of maternal genes by miR-430 shapes early development. Thus, learning about the function of miR-430 in zebrafish is likely to provide important insights into human development, cancer and cellular reprogramming.


Activation of the zygotic genome:

        Because a large fraction of the genome is maternally expressed, a significant challenge is to identify the first zygotically transcribed genes. We are developing methods to identify the first zygotic genes with the goal of defining their regulatory elements and ultimately identify the factors that mediate regulation of the zygotic genome activation.

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

The role of microRNAs in Vertebrate Development

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Introduction

        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. 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? To address these questions there are three projects open for postdoctoral positions: based on RNA Biochemistry, Developmental Biology and Computational Biology

Tools:


Zn finger and TALEN nucleases

We are using Zinc Finger and TALEN Nucleases to generate loss-of-function mutants in several zebrafish genes of interest.


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 Solexa sequencing

We are using high throughput sequencing and ribosome profiling to identify novel non-coding RNAs, characterize gene transcription and translation

GFP-miR-1 sensor repressed by muscle miR-1 and a control GFP transgene with the same promoter (below)

“how does an embryo develop from a fertilized egg”

miRNA ~30%

Y          >60%

zygotic

maternal

Zygotic stages:

activation of transcription

maternal mRNAs are degraded

Maternal stages:

transcriptionally silent

maternal mRNAs

Identification of a novel microRNA processing pathway:

Dicer is a central enzyme in miRNA processing. By analyzing mutants in Dicer and Argonaute2 we have identified a novel miRNA processing pathway that requires the catalytic activity of Argonaute2 instead of Dicer. We are currently undertaking high throughput sequencing of mutants in Dicer, Drosha, DGCR8 and Argonaute2 to identify novel non-canonical miRNA processing pathways, and annotate novel small RNAs in the developing vertebrate embryo

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 the mechanisms of miRNA function:

Several mechanisms have been put forward regarding miRNA mediated regulation of target mRNAs. We have used ribosome profiling (a technique developed by Nick Ingolia and Jonathan Weissman) to characterize the dynamics of translational repression as well as deadenylation and decay in the presence and the absence of miRNA mediated regulation. We have observed that for a large majority of targets, miR-430 first causes a reduction in ribosome occupancy, by reducing initiation rate and then it leads to deadenylation and decay.

We are currently applying this approach to define the translational start site for every zebrafish transcript,  characterize the post-transciptional regulation of mRNAs during embryonic development and annotate micropeptides, longer ORFs and lncRNAs in the genome.

Scheme of the Ribosome profiling technique (adapted from Ingolia et al Science 2009). Ribosomes are “frozen” in the message with cycloheximide and then the mRNA that is not protected by the ribosome is digested. After isolation of monosomes, the RNA is subject to high throughput sequencing, to characterize the fragments protected by the ribosome. in parallel the polyA+ RNA is purified and subjected to alkaline hydrolysis and seqeuncing to determine the input mRNA.

Post-transcriptional regulation by microRNAs:

The process of development requires a precise spatial and temporal regulation of gene expression. MicroRNAs (miRNAs) have recently been identified as widespread regulators of gene expression. miRNAs are ~21 nt small RNAs that repress gene expression post-transcriptionally. They constitute more than 4% of human genes and are predicted to target up to one third of all protein-coding genes.  These observations have uncovered an extensive layer of gene regulation that plays important roles during development and disease. In the laboratory we combine genetics with biochemistry and high throughput sequencing to characterize miRNA processing, mechanism of target regulation and function.