Epigenetic mechanisms and cell-type specific gene expression during anoxia and re-oxygenation in crucian carp brain

Crucian carp (Carassius carassius) is a fish that, unlike most other vertebrates, can survive for a long time without oxygen (anoxia). This ability is necessary for the crucian carp to survive in Northern Europe in small ponds, where the oxygen disappears when ice and snow prevent oxygen to enter from the air or photosynthesis. The fish survives anoxia with the help of a number of physiological adaptations. For example, lactic acid from glycolysis is converted to ethanol, which is washed out of the blood over the gills. In other vertebrates, the brain in particular is very sensitive to a lack of oxygen. Although we know quite a bit about how the crucian carp can physiologically survive without oxygen, there are still many questions that remain, especially for the brain.

We do not know how different parts and cell types in the crucian carp brain respond molecularly to anoxia. We will use a modern sequencing technique, ‘single-cell sequencing’, to see how much the different genes are expressed in single brain cells. This information can be used to identify different cell types and how they alter gene expression during and after anoxia exposure.

It is also unclear whether the molecular machinery and the necessary changes in gene expression are affected by changes in DNA methylation, a form of epigenetic regulation. We will investigate this with the sequencing technique ‘whole-genome bisulphite sequencing’. We will also investigate the enzymes involved in DNA methylation, as well as the presence and change in ‘long non-coding RNA’. This RNA affects the expression of other genes, and is another form of epigenetic regulation.

Anoxia tolerance is intriguing from an evolutionary perspective, but also important because oxygen deficiency is central to many biomedical issues. Like when you have a stroke, heart attack, or take out organs to save another human being. The research described here will form a necessary basis for future projects across life sciences and medicine.

The crucian carp (Carassius carassius) is a champion when it comes to surviving without oxygen, and much is known about the physiological mechanisms involved. However, molecular mechanisms regulating the physiological responses are unexplored. Preliminary research has indicated that many thousands of genes change their expression in the brain in response to anoxia and re-oxygenation. The magnitude of this transcriptional response has led us to hypothesise that the response may differ between cell types and brain areas, and may be orchestrated by epigenetic mechanisms such as differential DNA methylation. Furthermore, we hypothesise that specific long non-coding RNAs may also be differentially expressed and contribute to regulation of the response. Accordingly, the primary objectives are to investigate for the first time in crucian carp 1) differentially DNA methylated regions using whole-genome bisulphite sequencing, and 2) differentially expressed genes in different cell types and brain areas using single-cell RNA sequencing. We will also investigate the expression and activity of the DNA methylating enzymes and long non-coding RNAs, and we will examine genes and proteins resulting from the sequencing in further details using more standardised techniques. The proposed research itself addresses an important knowledge gap concerning the molecular mechanisms underlying the physiological mechanisms of anoxia tolerance. While the importance of epigenetic mechanisms in a variety of diseases as well as hypoxia are fairly well-established, there is no knowledge about their role in coordinating the physiological response to anoxia and re-oxygenation in crucian carp. Although it might be argued that anoxia is just an extreme form of hypoxia, it is important to bear in mind that there is a big difference between having a little bit of oxygen and having no oxygen, and knowledge from studies on hypoxia in other animals cannot be directly transferred.