Little RNAs - big roles?

Marika Kapsimali and Steve Wilson
Original paper reference
MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system

Our genome contains the instructions, in the form of genes, for making tens of thousands of proteins that are the building blocks and machinery of all the cells in our body. The genome is composed of DNA and each gene is a stretch of DNA that encodes a matching RNA that is then used as a template for building proteins. In recent years, it has become evident that not all RNAs that are encoded by the genome are used to make proteins. There are lots of very small RNA molecules called microRNAs or miRNAs that are thought to regulate whether other, larger, RNAs are made into proteins or are degraded. In this study, we aimed to examine the expression of lots of miRNAs in the developing and mature brain to find out which groups of neural cells might be using each of the different miRNAs. We reasoned that a detailed analysis of miRNA expression in different brain areas would be a useful resource for studies in the future that try to address the functions of the various miRNAs

Previous work in Ronald Plasterk's lab (Science 2005 309:310-1.) had identified a considerable number of miRNAs expressed in the zebrafish brain. For our study, we chose 38 that are conserved in vertebrates and have brain specific expression. A method called in situ hybridization was already established to detect their expression (Nat Methods. 2006 3:27-9.). The principle is to incubate cells with a labelled modified nucleic acid (Locked Nucleic Acid, LNA) that is complementary in sequence to the mature miRNA - if the miRNA is present in the cell, the complimentary LNA will bind to it and as the LNA can be visualised (the blue staining in the pictures below), one can determine which cells express the miRNA (the blue ones) and the ones that don't (pink in the pictures). Once the data on miRNA expression were gathered, we identified the brain areas where they expressed with the help of available neuroanatomical atlases (zfin.org).

Images of sections through the eyes showing a variety of expression profiles for different miRNAs. miRNA expression is detected by in situ hybridization (blue) and the sections are counterstained with nuclear red stain to visualize the cell nuclei. let-7b is expressed in proliferating cells (arrow, CMZ-ciliary marginal zone), mir-124 in the all differentiated retina cells, mir-183 in photoreceptors (Ph) and mir-181b in a subset of differentiating cells (INL-inner and GCL-ganglion cell layers).

The results show that miRNAs have a wide variety of different expression profiles in neural cells including: expression in proliferative cells and/or their differentiated progeny, regionally restricted expression, cell-type specific expression, constitutive expression in mature neurons. This suggests several modes of miRNA action in the neural cells. For example, cell-type specific miRNAs may modulate the spatial and/or temporal regulation of target mRNA translation within mature neuronal cells. Overall this survey provides an important resource for future functional studies of miRNAs in the brain.

Other links
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Scientific reviews
Bartel DP. Cell 2009 136:215-33. MicroRNAs: target recognition and regulatory functions.
Asli NS and colleagues. Curr Mol Med. 2008 8:698-710. MicroRNAs in organogenesis and disease.
Kosik KS. Nat Rev Neurosci. 2006 7:911-20.The neuronal microRNA system.

Six ways to make a brain

Gaia Gestri and Steve Wilson
Original paper reference:
Six3 functions in anterior neural plate specification by promoting cell proliferation and inhibiting Bmp4 expression

During development of the embryo, cells make decisions about the tissues they will contribute to and how they will differentiate. The outer layer of the embryo is called ectoderm and cells in this layer make the nervous system (brain and spinal cord) and the skin as well as migratory cells called neural crest. How do the ectoderm cells know which of these tissues to form? Within cells there are genes that encode proteins called transcription factors that act as molecular switches turning on different developmental programmes that control cell identity. In this study, we explored how one such transcription factor, called Six3, promotes the ability of cells to form eyes and brain.

Six3 is sometimes called a 'master regulator' for eye and forebrain development in that it is necessary and sufficient for the eyes and brain to form. What this means is that in gain of function experiments (in which the embryos express more Six3 protein), embryos show brain enlargement and ectopic eye structures (see the arrow pointing to 'the third eye' in Fig.1) while in loss of function experiments (where the Six3 protein is absent or not functioning) embryos show reduction of the most anterior part of the brain and no eyes (Fig. 1, right).

Figure 1. Pictures of the heads of frog tadpoles the black blobs are the forming eyes. In the tadpole on the left there is a small extra eye on top of the head when there is too much Six3 protein and the tadpole on the right is missing an eye due to lacking Six3 protein on one side of its brain.

So how might Six3 control brain and eye development? With this in mind, we asked when and how Six3 exerts it function taking advantage of both the frog, Xenopus laevis, and the zebrafish as model systems for our experiments.

The processes that regulate brain development start at the early neural plate stage, this is much earlier than the first morphological appearance of the eye and the brain. The neural plate is a sheet of cells that will form both the brain and the eyes and at first, it is continuous with a sheet of cells called the epidermis that will form the skin. It is the anterior part of the neural plate, ANP, (Fig. 2 red) that gives rise to the brain and eyes (Fig. 2).

Figure 2. The schematic shows a very young frog embryo where the cells that are going to form the nervous system are coloured in red and blue. The red cells are those that will develop into the brain and eyes (right photo)

Given that the ANP has the capacity to form the brain and eyes, then the key genes that control eye and brain development must already be active in the ANP. These ANP genes must positively confer brain/eye identity as well making sure that the cells don't acquire the identity of the surrounding tissues such as the posterior neural plate (blue in Fig. 2), that will give rise to the spinal cord, and the surrounding epidermis that will give rise to the skin (brown in Fig 2). In our study we found that Six3 plays key roles in both specification and maintenance of the brain/eye promoting properties of the ANP.

Figure 3. Schematic and two photos of the anterior neural plate (blue in the schematic and white in the two photos. The neural plate is surrounded by epidermis (yellow in the schematic and blue in the photos). The little brown dots in the photos are individual cells that either have a lot of Six3 (middle), or lack Six3 (right)

One of the properties of the ANP is that it is more highly proliferative compared with the posterior neural plate and we found that Six3, expressed in this territory, promotes proliferation and represses the production of neurons.

The ANP is surrounded by the epidermis, (Fig. 3) that will later form the skin. The epidermis produces a signal, called Bmp, that has the ability to inhibit expression of ANP genes. We found that Six3 switches off Bmp expression (Fig. 3 middle) ensuring the maintenance of the ANP as a Bmp-free domain. This is really important given that if Six3 function is lost, Bmp signalling encroaches into the ANP (Fig. 3 right) and doing so, leads to embryos with severely reduced anterior brain and eyes and expanded epidermis (Fig. 1 right panel).

In humans, mutations in Six3 can lead to severe congenital abnormalities of the brain and eyes, and this study has helped to elucidate the function of this gene, one of the most important genes for brain and eye development.

This study was done as a collaboration between Guiseppina Barsacchi's lab in Pisa and Steve Wilson's lab here at UCL.