Week 2 Diary

Day 6

Over the weekend the chick embryos have been on “the shaker” in the cold room to ensure that the antibody has well and truly mixed with the embryo and managed to penetrate into the cells to bind with any of the chemical on the nucleotides.

20130722-184817.jpg
“The Shaker”

The rest of the practical work for the day involved rinsing in MABT once every half an hour, to remove any unbound antibody. Consequently, there has been lots of time for discussion about the next part of the project. We are going to look at the genes that are expressed before the ones we are currently examining. These are genes that code for transcription factor proteins. A transcription factor controls which genes are expressed and when. In effect they turn genes on and off. So the genes that code for neuron transcription factors will be expressed in the messenger RNA before the genes that then cause (neuron induction (determining which cells are to become neurons) and neurogenesis (the differentiation of these pre-neural cells into neurons). This means that the transcription factors are made first, which then switches on the genes for neuron differentiation, so that these proteins are made. The transcription factor genes that we will look at are neurogenins 1,2 and 3.

We will use many of the same techniques as before, exept we will need to clone these genes before we can make the probes, in order to produce enough to work with. We will then be able to produce images of the stained cells indicating the expression of the genes, which we will add to our timeline of expressions of all the genes we will have examined. So, what we expect to see is that the neurogenins are expressed first, then the genes that we are currently examining, including Neuro D and M, Sox 2, Notch 1, Cash 1 and Crabp1, with Crabp1 somewhere in the middle. But, we will have to wait for the final photographs to find out…

Day 7

Today was results day where we could finally see if my in situ hybridisation work (adding the probes, then the antibody then the substrate stain with lots of washing in between) had been successful. When we added the antibody before the weekend, it specifically bound to any of the probes that had bound to the messenger RNA containing the gene. This antibody had an enzyme attached to it, so the enzyme too became bound to any of the messenger RNA. So, when we added the substrate today, it entered the cells and was converted into a blue stain by the enzyme, but only if the enzyme was present in the cell. The only way this could be was if the enzyme had become bound to the messenger RNA, because we had washed the chick embryos so many times that there was very little unbound antibody-enzymes left. In this way, all the cells containing the activated gene in their messenger RNA would be stained blue.

A few hours later and it became clear that almost all the chick embryos show some staining, in different regions and in different intensities. This is great news because first, the washing worked and more importantly, we have good data to construct a timeline of the activation of the genes and we will be able to produce numerous photographs. These will act as evidence for the locations and activations of these genes in neuron development which will provide a basis for understanding their roles. For example, if two different genes are activated at the same time but in different places, it may be the case that they are both involved in neurogenesis but for different types of neuron cells.

It is rewarding to see my practical work come to life, and to have produced some good results when there was the possibility that many may have been unsuccessful. The precision of each stain is particularly interesting because in areas where the staining is less intense, each stained cell appears as a tiny dot and it is possible to see individual cells where the genes have just switched on. There will be pictures up soon!

Day 8

Photography day!

Now the washing is finished, the next stage is to put the chick embryos in glycerol, where they can be stored for up to 20 years! But that was all the preparation for today. After this, they could go straight under the microscope and we would get a better look at the staining. This is the really interesting part.

Before I took the photos of the results the staining, I examined each chick embryo under the microscope. This provided greater detail of the staining and of the chick embryo structures than I could see whilst I was washing them. Comparing the staining achieved through the use of different gene probes, at different stages made the sequence of the activation of the genes much clearer. So, for example, at stage 10, the chick embryos tested with Crabp1 showed, under the microscope, very little staining, whilst the Notch1 and Sox2 chick embryos have very intense staining.

This may suggest two things. First that Crabp1 is activated later in development and second that it is activated in fewer neuron cells so may be a marker of a specific type of neuron cell. We know already that Notch 1 is involved in Notch signalling which determines the cell fate of ectoderm (a germ layer) cells, ie whether they will become endodermis or neuron cells. This means Notch 1 will be activated much earlier, at the very start of neuron differentiation, in the neuron induction stage, and in all ectoderm cells.

This knowledge of the chick embryo structures and a rough idea of the location of the staining was useful when photographing the chick embryos under the microscope. I knew which areas were important to focus on with the camera to demonstrate the gene activation sequence for my results paper.

By the end of tomorrow, I hope to have finished photographing all the chick embryos, and will have some to post on the blog.

Definitions:
Germ layer: A layer of cells in embryo development. The embryos of most species have three germ layers, the ectoderm, mesoderm and endoderm, each of which lead to the development of different cell specialisations.

Ectoderm: The germ layer which differentiates into neurons or endodermis.

Day 9

Although I said yesterday that I hoped to have finished the
photographs, in reality, I am about half way through and already I
have half a gigabyte of photos! The microscope I have been using today
has a very high resolution giving very good photos, but which take up
lots of memory! This microscope has a very precise focus and allows
me to examine each (transparent) layer of the chick cmbryo. So for each chick embryo, I took a photo of several cross sections, without actually having to cut through anything.

The pictures show some great detail, particularly in the stained
cells. Because these are coloured, they can be distinguished
individually with clear nucleii. Nucleii don’t stain because the
nucleus contains too little messenger RNA to give an intense purple, as
the rest of the cell does.

For these pictures, I mounted the chick embryos on slides. They
need to be dorsal-side up to get the best view of the developing
nervous system, which takes some patience to work out. Examining stuctures at this magnification, I have learned a lot of surprising and seemingly random things from the project so far, the unrecognisable anatomy of early chick embryos being
one of them.

I am building a good set of results, though they are differrent from the sort of results that I am used to getting in school science experiments. Rather than numerical data, which can be rather meaningless until presented visually for example in a graph, this data is already visual. The analysis will involve photographs rather than graphs and figures.

I will post the photographs once I return from my holiday, along with more diary blogs as I continue the project.

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