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There are several projects currently ongoing in the laboratory, with the main goal of understanding what are the functions of microRNAs during vertebrate development.


Regulation of the maternal-to-zygotic transition by microRNAs.


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 the year 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. 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 and cancer.

 

The role of microRNAs in Vertebrate Development

Introduction

In the Giraldez’ lab we use zebrafish as a model system to investigate the role of microRNAs during vertebrate development. We combine genetics, embryology, genomics, chemical, and computational biology to address a central question in biology: how does a fertilized egg develop into a complex multicellular embryo? This process 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. While these observations have uncovered an extensive layer of gene regulation that plays important roles during development and disease, their specific functions are still unknown.

To investigate the role of miRNAs, we have generated embryos devoid of miRNA processing by eliminating all dicer activity (maternal-zygotic dicer mutants, MZdicer) (A, B). (C) By injecting microRNA duplexes in MZdicer, we can investigate the function of a  single miRNA in the absence of other miRNAs. We have identified miR-430, an abundant miRNA expressed during early zebrafish development and conserved in other vertebrates. (D) Providing miR-430 in MZdicer rescues early development, highlighting the importance of this miRNA during the early developmental period.

The role of microRNAs during brain morphogenesis:


During vertebrate development, the neural tube undergoes morphogenetic movements to form the forebrain, midbrain, hindbrain, brain ventricles and mid-hindbrain boundary. MZdicer mutants fail to undergo brain morphogenesis, but injection of miR-430 in the early embryo is able to rescue brain morphogenesis and mid-hindbrain boundary formation. These results suggest that miR-430 plays important roles in the regulation of gene expression within the neural tube to allow brain morphogenesis. We are currently studying the miR-430 targets and how their regulation instructs cellular movements during brain development in vertebrates.

Identification of small molecules that regulate miRNA function:


microRNAs have recently been involved in a variety human diseases, including, muscular dystrophy, Tourette Syndrome, and cancer and tumor metastasis. The identification of small molecules that interfere with the processing and the activity of miRNAs might yield important therapeutic agents to treat human diseases. To this end we have developed in vivo and in vitro studies to identify small chemicals that interfere with the miRNA pathway. These compounds will hold great therapeutic potential to treat human diseases where miRNAs play important roles.


Identification of novel genes involved in the miRNA pathway:


Using mutagenesis in zebrafish, we are undertaking a genetic screen to identify mutants that affect miRNA function, as a means to discover novel components in the miRNA pathway.

Tools:


Zn finger nucleases

We are using Zinc Finger 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.


microarray analysis

We are using microarray analysis to identify miRNA targets in vivo, by analyzing mRNAs upregulated in the absence of miRNAs.


High throughput Solexa sequencing

We are using high throughput sequencing analysis to identify novel small RNAs

Role of microRNAs during organogenesis:


To investigate the role of miRNAs during organogenesis, we are using two complementary approaches: First, we are using MZdicer mutants and single miRNA inhibition to identify the cellular processes affected by the absence of miRNAs. Second, we have observed that miRNAs accelerate target mRNA degradation. Thus, microRNA targets can be identified by looking at mRNAs upregulated in the absence of microRNAs. We are using microarray analysis in dicer mutant embryos to identify tissue-specific microRNA targets in neurons and muscle cells. Combining in vivo target identification with the phenotypic characterization of dicer mutants will help us to understand the function of tissue-specific microRNAs during cell fate specification and tissue homeostasis.