Sequencing most fowl: a poultry challenge

Mark Tizard and colleagues have recently had a paper accepted for publication in Genome Research that catalogues a substantial list of microRNA (miRNA) sequences from the chicken. Not so exciting you may think, but it actually provides a valuable new resource for researchers working with chickens either as a model system for vertebrate development or as a production species.

Assembling the catalogue also ticks off the first box in an ambitious project being led by Tizard, a senior scientist based at the Australian Animal Health Laboratory (AAHL) in Geelong, Victoria, to characterise the roles of miRNAs in chicken development and physiology. The project is a joint effort between CSIRO Livestock Industries, Plant Industry and Entomology as part of a CSIRO 'Emerging Science' initiative called Cellular Reprogramming.

miRNAs are single-stranded RNA molecules of about 18-24 nucleotides in length that are important in regulating gene expression. It is now known that these molecules use RNA interference pathways. Tizard's miRNA project grew out of research on applying RNAi technologies in livestock therapeutics, led by fellow CSIRO researcher Dr Tim Doran (see ALS May/June 2007). Specifically, the group is using RNA interference to target avian influenza.

By way of background to his own research, Tizard recalled how excited Tim Doran was when the first lot of work describing the RNAi mechanism started appearing several years ago, including the groundbreaking, Nobel Prize-winning papers by Fire and Mello.

At the same time, similar observation were being made in plants closer to home by Dr Peter Waterhouse and Dr Ming-Bo Wang at CSIRO Plant Industry in Canberra, who developed patented DNA-delivered (dd)RNAi technology based on their observations of RNAi in plants.

Doran immediately saw the potential of this previously unrecognised level of gene regulation via blocking mRNA translation, and was keen for CSIRO Livestock Industries to join in. The applications were immediately apparent, including trait modification and disease prevention. "It was around that same time that microRNAs were being uncovered," Tizard says. "DNA sequences that affected the timing of morphology changes were identified in C. elegans. These turned out to be tiny fragments of RNA nowhere near big enough for a coding sequence ... miRNAs.

"Now, CSIRO Livestock Industries has invested a lot of money over the years in trying to understand animal genetics and how the genetics is delivered. Here was evidence of a whole new layer of control in the cell for gene expression, and with that, effects on animal growth and development. So, with Tim's RNAi project up and running four or five years ago, it became obvious that we should be also looking at miRNAs in our systems. I was lucky enough to be in the right place and the right time to start this project."

The team chose the chicken as the model system for the miRNA project for several reasons. Firstly, the group at Geelong had a lot of experience with the chicken as a model organism and therefore many of the tools were already in place.

Secondly, the chicken is a fundamental model for studies of vertebrate development, which has importance for CSIRO interests and the wider research community. In particular, they are trying to better understand development of the immune response in the chicken production environment and how to enhance that immunity so that the animals remain healthier during production and more resistant to pathogenic attack, which usually viral.

This particular focus of course relates to the broader CSIRO project on avian influenza. And finally, from more of a pragmatic viewpoint, scientists can work on chicken embryos without having to go near the living animal.

"Chickens pump out these lovely sterile packages that act as a critical resource for our experiments," Tizard says. "This means we can study embryo development with precision at early stages without going near the mother.

"This is a major consideration in terms of the animal welfare and animal ethics requirements for research in this day and age. We are not impacting on the grown bird at all."

---PB--- miRNA-omics

Besides having relevance in the wider research community, the chicken is also an end-point organism for CSIRO as a significant livestock production animal. Tizard says that it is also likely to become even more important in the future because it is a source of high-quality protein/meat, but has a vastly reduced impact in terms of environmental footprint for production compared to some other production animals.

"Even at the free-range end of things, chickens take up less space and need less food and water per kilogram of meat generated and everything is quicker as well with an average production lifespan of 35-45 days," he says.

In terms of the global animal biotechnology market, the chicken is also valuable. The rate of stock turnover means that delivery of high-quality genetics to the marketplace is relatively easy.

The reported miRNAs in Tizard's Genome Research paper brings the number of miRNAs for the chicken in the Sanger miRBAse from 149 to 445 (including 19 new homologues and 277 novel sequences), with an additional 138 miRNA candidates to be validated.

Not unexpectedly, the highest number of annotated miRNAs in the Sanger database is in humans, at 678, with the actual number predicted to be much higher. For the mouse, the number is 472, and for zebrafish, 337 sequences. So, the chicken is right up there.

This miRNA catalogue gives Tizard's group an idea of exactly what they are dealing with miRNA-wise, or more precisely what the chicken is dealing with. The next step is to work out what all these miRNAs are doing in the cells.

Tizard's team is now characterising tissue-specific expression patterns using microarrays and in situ hybridisation to assemble a map of normal miRNA expression in different cells and tissues at different developmental stages.

"We've also got some leads on interesting differential processing of miRNAs, which is an emerging area in terms of understanding the mechanisms: that is, controlling the controller."

The plan then is to apply their findings to a range of areas. In the health arena, Tizard is interested in development of the immune system and in particular cells of lymphocytic lineages. "We would like to know which miRNAs are involved in determining the fate of the CD4 and CD8-type T cells because in the modern production environment for chicken, viral diseases are the greatest threat and of course the greatest of these in terms of public awareness is that of avian influenza."

Tizard's studies of miRNAs will also fit in with wider studies of gene expression going on in other parts of CSIRO. "In any study of transcriptomics, I think you have to now consider the miRNAomics, so that you can get a handle on which messenger RNAs are going to be affected by miRNA expression."

---PB--- Applications

In terms of production applications of the miRNA technology, the ideal outcome would be some sort of switch or control. "If a particular miRNA is expressed in a specific pattern, we want to be able to increase or decrease it, or even switch it off or on," Tizard says. "Basically we are looking for common or key regulatory pathways to target in the production environment."

In a broader application, Tizard's team also collaborates with Professor Andrew Sinclair and Dr Craig Smith at the Murdoch Childrens Research Institute in Melbourne to study sex determination. Together they are trying to establish how miRNAs might be involved in the process.

"This is a really major cellular reprogramming event that changes an embryo's fate with major anatomical and physiological differences in eventual phenotype," Tizard says. "From a production viewpoint, it becomes fairly obvious that female chickens are necessary for egg production, and males are better for meat production.

"So, a switch to control germ cell fate would be potentially beneficial. Obviously, Andrew's group is interested in how gender is determined during development and what goes wrong with it, especially in human, then how miRNAs might be involved. Our work is generating a suite of tools to address those questions."

Another interesting aspect that has emerged from the miRNA project, according to Tizard, is how it is feeding back into the parent project on RNAi.

"It has become clear to us that while you can bring about RNA interference by dumping dsRNA on cells and letting them deal with it, it is perhaps more efficient and biologically relevant to express the sequence that you want in the cell and use the cell's machinery to process those transcripts - that's the concept of hairpin RNA expression, or ddRNAi - and that's the method that Tim's group is using.

"That process essentially mimics what miRNAs are doing. Therefore understanding how miRNAs are made, their biogenesis, how they are pushed out into the cytoplasm, which ones are processed most efficiently, etc you begin to get a handle on the kinds of structures and sequences involved in promoting that, and you then open up the possibility of tailoring better constructs for RNA interference, be it for antivirals or trait modification or whatever."

In the short term, Tizard is looking to build on the current findings in their miRNA work, and to identify a clear application for CSIRO's external stakeholders. They have some candidate miRNAs in the pipeline with the potential to alter lymphocyte differentiation, and future results in the sex determination work will clearly have the potential attract external research support.

"We are also interested in the viral infection area and how the changes in gene expression relate to levels of miRNA expression during infection with avian influenza, in particular."

Coming from a basic research background before joining CSIRO a decade ago, Tizard is occasionally torn between the pull of pure discovery and the focus on outcomes for external stakeholders.

However, he admits to being in a good situation. "We are lucky in that the research management here at CSIRO has always promoted the idea that if you keep your eye on doing the fundamental science and addressing an important problem, it will reveal an important application. It is all about the question and how you go about addressing it."

(Tizard's group includes two postdoctoral fellows, Pauline Cottee and Evgeny Glazov and a PhD student, Stephanie Bannister. He works closely with other senior scientists, Rob Moore, Brian Dalrymple and Tim Doran at Livestock Industries together with Chris Helliwell and Frank Gubler at Plant Industry.)