POSTER TEMPLATE BY:

www.PosterPresentations.com

Genes involved in the regeneration of dopaminergic neurons in zebrafish larvaeMatthew Guo, Rafael Godoy, Sandra Noble, Marc Ekker

Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa.

AbstractZebrafish has a special characteristic that allows them to regenerate body organs in response to injury, a phenomenon which also includes regeneration in the CNS. The dopaminergic (DAergic) neurons of transgenic zebrafish Tg(dat: CFP:NTR) were ablated upon exposure to metronidazole (Mtz) treatments. This ablation method is a successful and efficient model for loss of DAergic neurons which are seen in Parkinson’s disease (PD) patients. We observed the changes in transcription of several genes that have roles in neurogenesis, or involved in the regeneration of damaged tissues, which we hypothesize to play a role in the regeneration of DAergic neurons in the brain. The identification of these changes further reinforces the idea that a regenerative stimulus takes place following DAergic neuron ablation. By investigating these changes and understanding how DAergic neurons regenerate in the larval and adult zebrafish, we hope to develop novel therapeutic approaches that may aid in human neurodegenerative diseases such as PD.

Introduction

Methods

Results

Results

Future Directions

• Dopamine (DA) is a neurotransmitter involved in learning, behavioural responses and regulation of motor abilities (Noble, AJMG.b 2003). Decrease in the number of dopaminergic (DAergic) neurons in the substantia nigra par compacta leads to the development of Parkinson’s disease (PD) (Lotharius et al, NRN 2002). PD is the second most prevalent neurodegenerative disease in people over the age of 55 (Dextera et al., j.freeradbiomed 2013), and there is no cure due to its unknown pathology (Klein et al., ncpneuro 2006).

• Unlike humans, zebrafish has the capability to regenerate neurons in the CNS (Gage et al., j.neuron 2013). We investigate the involvement of a number of genes that are known to play a role in embryonic neurogenesis in zebrafish to look for potential roles also involved in DAergic neuron regeneration. In addition, genes involved in the response to cellular injuries and genes involved in providing support and protection to the neurons are also studied.

• Using a transgenic zebrafish line developed in the Ekker lab, DAergic neurons are ablated and the expression levels of various genes are compared to the untreated control group. This may assist in the understanding of which exact molecular mechanisms are being activated during neuroregeneration in zebrafish, and therefore assist in the discovery of novel targets for the treatments of PD.

• Genes of interest include AXIN2, BDNF, GATA3, GFAP, Nestin, Neurogenin1, OLIG2, PCNA, PITX3 and SHHa.

Conclusions

0 dpf

Time

1 dpf

Dechorionation & Mtz/DMSO

treatment

2 dpf 3 dpf 5 dpf

Sacrifice

IHCqRT-PCR

Mtz 5mM Mtz 7.5mM

4 dpf

Mtz 7.5mM

45 dpf

Sacrifice

ddPCRFigure 1: Experimental timeline of Mtz treatment

Figure 2: Schematic of NTR-Mtz cell mediated ablation Figure 3: Mtz administration results in loss of dopaminergic neurons in the Tg(dat: CFP-NTR) line

CFP TH Merge

Figure 4. Fluorescent images of 5 dpf zebrafish head stained for PCNA

Figure 5. qRT-PCR data of gene expression fold-change

Figure 6. ddPCR data of absolute cDNA concentration (copies/μL) of genes

Brain Brain Brain Whole fish Whole fish Sample 1 Sample 2 Sample 3 Sample 1 Sample 2

10

5

0

-5

-10

Figure 7: Heat map of gene expression fold-change

• The NTR-Mtz ablation system is an effective method to globally ablate DAergic neurons in a spatial, temporal and controlled manner.

• By using different methods to observe changes at the RNA and protein levels, we have identified ten genes which may be important for the neuroregeneration process seen in zebrafish.

• Further research to study the activity of promising genes such as AXIN2, PCNA, GATA3, and Shha is required to understand the implications of these genes in the zebrafish brain regeneration process.

• Conducting experiments to observe the changes at the protein level (such as IHC and western) during regeneration will add crucial information towards understanding mechanisms involved in neuroregeneration.

• in vivo lineage tracing of neurons may be utilized to observe where the proliferative cells migrate to and confirm if they do differentiate to DAergic neurons.

Vehicle Mtz