Investigation of Syntaxin 3B in Developing Zebrafish EmbryosDerek Anderson* and Wendy Boehmler, PhD

Department of Biological Sciences, York College of Pennsylvania

Objectives

• Successfully isolate the gene encoding syntaxin 3B from the zebrafish retina

• Determine the expression of the syntaxin 3B gene in adult zebrafish gene

• Investigate the temporal expression of the gene throughout the development of zebrafish embryos

MethodsRNA

Isolation

cDNA Synthesis

PCR for the location of syntaxin 3B

PCR for the time points of gene expression

Clone gene into pDRIVE vector

Results

Figure 1. Agarose gel electrophoresis from a PCR of cDNA collected from adult zebrafish (Danio rerio) tissues. Tissues tested were the eye (E), heart (H), muscle (M), gut (G), and brain (B). No cDNA was used with the negative control (C). Visualization of DNA was done with Ethidium Bromide staining. Syntaxin 3B appears to be expressed in only the retinal tissue of adult zebrafish.

Results

Figure 2. Agarose gel electrophoresis from a PCR of cDNA collected from developing zebrafish (Danio rerio) tissues. Embryonic stages were 24hpf, 48hpf, and 72hpf. Visualization of DNA was done with Ethidium Bromide staining. Syntaxin 3B appears to be expressed in the 24hpf and 72hpf embryos, but little to no expression in the 48hpf embryos.

Table 1. Syntenic relationship of human and zebrafish genes. The chromosomes in humans and zebrafish containing syntaxin 3 share an additional eight genes. This supports an evolutionary link and that the gene we isolated is the syntaxin 3 homolog. (Woods et al. 2005)

Gene Name Zebrafish Chromosome Human Chromosome

syntaxin 3gstp1

mgc13045syvn1

polr2glloc254359loc51035

clp1req

141414141414141414

111111111111111111

Figure 3. Comparison of human, rat, mouse, and zebrafish syntaxin 3 genes. Human, mouse, and rat syntaxin 3 genes were aligned to the zebrafish syntaxin 3B gene using ClustalW. Dashes in sequences allow optimal alignment for amino acid insertions or deletions. Identical amino acids are highlighted by asterisks, while highly conserved amino acids are highlighted by dots. The SNARE domain of syntaxin 3 is highlighted in yellow, while the transmembrane domain is highlighted in teal.

Literature Cited• Curtis, Leigh, Proletta Datta, Xiaoqin Liu, Nataliia Bogdanova, Ruth Heidelberger, and Roger Janz. 2010. Syntaxin 3B is essential for the

exocytosis of synaptic vesicles in ribbon synapses of the retina. Neuroscience. 166(3): 832-841.

• Curtis, Leigh B., Blair Doneske, Xiaoqin Liu, Christina Thaller, James A. McNew, and Roger Janz. 2008. Syntaxin 3B is a t-SNARE specific for ribbon synapses of the retina. Journal of Comparative Neurology 510(5): 550-559.

• Ming Li, Wai, Sarah E. Webb, Karen W. Lee, and Andrew L. Miller. 2006. Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos. Experimental Cell Research 312: 3260-3275.

• Woods, I.G., Wilson, C., Friedlander, B., Chang, P., Reyes, D.K., Nix, R., Kelly, P.D., Chu, F., Postlehwait, J.H., and Talbot, W.S. 2005. The zebrafish gene map defines ancestral vertebrate chromosomes. Genome Research 15:1307-1314.

Future Studies

• Determine the spatio-temporal expression of Syntaxin 3B in developing zebrafish embryos using whole mount in situ hybridization

• Examine the functionality of the gene by developing knockout models of zebrafish for Syntaxin 3B

Acknowledgements

I would like to thank Dr. Boehmler for all of her help throughout this project.

Conclusion

• Using PCR, it was determined that Syntaxin 3B is present in the eye tissue of zebrafish

• Using PCR, it was determined that Syntaxin 3B is present in developing zebrafish embryos at 24hpf and 72hpf.

• Syntaxin 3B is located on chromosome 14 of zebrafish and 11 in humans. These two chromosomes share many similar genes demonstrating synteny.

• This gene is also highly conserved between species.

Zebrafish Syntaxin 3B Gene

Forward ReverseCDS: 696 bp

Curtis et al. 2010

http://zf-health.org/information/factsheet.html

Introduction

• Ribbon synapses are located in the retina and in hair cells of the ear

• Ribbons are specialized for continuous neurotransmitter release

• In order for a neurotransmitter to be released from the presynaptic terminal, a SNARE complex fuses the vesicle loaded with neurotransmitter to the plasma membrane for exocytosis

• One of the proteins making up a SNARE is syntaxin (found on the plasma membrane) which interacts with synaptobrevin (a protein found in the membrane of the vesicle)

• Although the family of syntaxins are located throughout the nervous system, syntaxin 3B is specifically expressed in retinal bipolar ribbon synapses

• At this time, little is known about its role in development