Hello. Welcome back to our lecture in microRNA epigenetics. And in section D, we will talk about the role of microRNA in organism development and, in particular, in neural differentiation. MicroRNA's expression patterns are tissue and organ-specific. Some microRNAs are expressed solely in the brain like mir-9 and mir-124. Some microRNA are enriched in the lungs, for example, mir-224. Mir-122 microRNA is expressed only in liver, and this is liver-specific. Mir-206 is expressed in skeletal muscles, while mir-1 is expressed in heart. MicroRNA expression is not only spatial specific but also temporal specific. That means that during the development or the cell differentiation, the microRNA expression patterns are changing. On this slide, you can see northern blots, what I have been doing during my PhD, where you can see the induction of expression of let-7 and mir-125 during mouse brain development in section A. E12 through E17 is embryonic days 12 and 17, and P0 is postnatal day zero. We could see induction of those microRNAs not only during the brain development, but also doing the differentiation of embryonic stem cells. We differentiated mouse embryonic stem cells into neurons, as you can see in panel B. And we found that neural-specific microRNA such as, example here, let-7 and mir-125, were not expressed in undifferentiated stage. However, they are strongly induced 12 days after induction of differentiation. Moreover, in their context on the brain, microRNA expression was cell type-specific, as you can see in section C, where we compared the expression of several microRNAs and neurons and astrocytes. In majority of microRNAs were very specific for neurons and were not expressed in the astrocytes. And, for example, microRNA such as mir-23 was highly expressed in astrocytes, but not in neurons. So does it mean that microRNA are specifically expressed in different stages of embryonic brain stem cell differentiation and they regulate their differentiation process? Do they regulate switch from one stage of development to another? Do they regulate the switch from one lineage to another? And this type of questions, we and others, are basing their research to study the changes in microRNA expression and their role in differentiation from pluripotent stem cell stage to the neurons and astrocytes. As [inaudible] explained in the previous session where he described how the microRNAs were discovered, in C. elegans, the reciprocal expression of microRNA targets and microRNA during development, regulate the transition from one larval stage to another into adult. The same can be found in the vertebrates. The same microRNAs, let-7 and lin-4, in invertebrates, homologue mir-125, are induced during differentiation and block their genes which are highly expressed in early stage of differentiation, as you can see in the lines in the B panel of this figure. The left panel of the slide shows you the concert reciprocal temporal expression of two microRNAs, lin-4 and let-7. In the upper panel you can see that these two microRNAs are induced during the development of C. elegans in later stages and while the lin-14, lin-28 and lin-41 genes are going down. The same happens in invertebrates where in early stages in undifferentiated stages of embryonic stem cells, lin-28 and lin-41 is highly expressed in the course of differentiation. They are downregulated by let-7 or mir-125, which are highly expressed in later stages of differentiation. On the right panel of the slides, you can see so-called double negative feedback loops between microRNA let-7 and pluripotent C markers lin-28 and lin-41. Basically, in pluripotent stem cells, the lin-28 is highly expressed and is regulate let-7 processes. However, during the differentiation, this balance between lin-28 and let-7 shifted into two let-7 expression. Let-7 is highly expressed in differentiated cells and inhibit lin-28. This cartoon summarize the role of microRNA in neural development. Basically, it's not only two microRNAs, lin-1 and 7, which regulate the differentiation. About 30 percent of all known microRNAs are expressed in the brain where they perform fine tuning of neurogenesis, regulating neural development, brain morphogenesis and neural stem cell differentiation. Mir-124 and mir-9, this is two microRNAs which are most abundant in the brain and they regulate the transition from neural stem cell stage to mature neurons. Cartoon to the right shows there are two different stages of neural stem cell differentiation through neural stem cells, neural progenitor cells and to the mature neurons. The expression of microRNA during neural system development is temporally regulated. Some, like let-7 and mir-125, are expressed already in neural stem cells and the others in progenitor cells. However some of the microRNAs are specific for mature neurons. MicroRNA regulate the neural differentiation interplay with other factors. Very often, microRNAs and transcription factors are involved in so-called regulatory negative feedback loops, as it's shown on this figure for two examples, mir-124 and transcription factor SOX9 and mir-9 and transcription factor TLX. I will describe the feedback loop between TLX and mir-9, as example. So, in neutral stem cells, TLX transcription factor is highly expressed and it's regulate the self-renewal and proliferation of the stem cells by blocking mir-9. However, in course of differentiation again, the balance between TLX and mir-9 expression is shifted towards mir-9. Mir-9 is induced, and through this induction, it's repressed TLX expression and cells become neurons. In this section, I introduced you to the most important role of microRNAs in regulating developmental timing on the example of neural development and stem cell differentiation. The microRNA play a major role.