Gene Expression Across Different Stages of Regeneration in Amphiura filiformis

SHANA HARDY

Objectives of the Study• Previous studies: SINGLE gene studies• This study: MULTIPLE gene study• 3 stages of regeneration• 3 groups of genes of particular interest

• Hox gene regulation• Neuronal development• Bone morphogenic protein-1 (BMP-1)

Why use Amphiura filiformis?

• Can live in extreme temperatures (tropical/arctic)

• Share 70% of genome with humans (Sea Urchin Genome Sequencing Consortium, 2006)

• Can regenerate adult organs

http://www.marlin.ac.uk/assets/images/marlin/species/web/o_ampfil3.jpg

3 Phases Being Studied

• Cell proliferation in undifferentiated blastema

• 7 days post autotomyBlastema

Stage

• Both cell proliferation and differentiation

• 3 weeks post autotomy

• 50% differentiated

Intermediate Regenerative Phase (50%

DI)• Some

proliferation in the distal part of arm

• 5 weeks post autotomy

• 95% differentiatedAdvanced

Regenerative Phase (95%

DI)Figure 1. Amphiura filiformis regenerated arms at different stages of differentiation.(A)Blastema, 7 days p.a. (B) 50% differentiated, 3 weeks p.a. and (C) 95% differentiated, 5 weeks p.a.Burns, Gavin, Olga Ortega-Martinez, Michael C. Thorndyke, Lloyd S. Peck, Samuel Dupont, and Melody S. Clark. "Dynamic Gene Expression Profiles during Arm Regeneration in the Brittle Star Amphiura Filiformis." Journal of Experimental Marine Biology and Ecology 407.2 (2011): 315-22. Web.

Animal Collection and Sampling

• 4 replicates• 15 animals• 30

regenerates (~2 arms/animal)Blastema phase

• 4 replicates• 15 animals• 30

regenerates (~2 arms/animal)

Intermediate Regenerative

Phase • 4 replicates• 15 animals• 30

regenerates (~2 arms/animal)Advanced

Regenerative Phase

In total: 180 animals in experimental group

40 animals in control group

Methods used:

1. cDNA library production and microarray fabrication

2. RNA and target preparation for microarray analysis

3. Data analysis (ran microarray analysis)

4. Clone sequencing and analysis

5. Quantitative PCR measurement of gene expression

Picture source: http://www.microarray.lu/en/MICROARRAY_Overview.shtml

Results of the Microarray

• What is a SOTA cluster?• Self Organizing Tree

Algorithm• What is cluster analysis?

• Method of classification through use of dendograms as a way of categorizing data based on the similarity of the data points to one another (Schena, 2003)

• What does this dendogram mean?• Log 2 ratio > 0 = increase• Log 2 ratio < 0 =decrease

Figure 3. Table describing the number of clones in each SOTA cluster and a SOTA dendogram showing the average profile of the clusters (Burns et al., 2011)

Results From Microarray vs. Quantitative-PCR

From Figure. 4: Comparison of microarray (red) and Q-PCR (blue) results (Burns et al., 2011)

Discussion of Differentially Expressed Genes Across the 3 Regenerative Stages

Early Regenerative Stage

• Mostly involved in proliferation and energy production

• ATP synthase• Cytochrome

oxidase• Ribosomal

proteins• Elongation

factors

Intermediate Regenerative Stage

• Similar gene expression profile to blastema stage

• Chaperonins• Polyubiquitin• SOX-1

Advanced Regenerative Stage

• Overall decrease in gene expression levels

• BI-1: may be related to slowing down the rate of cell division as cells become fully differentiated

Discussion of Differentially Expressed Genes Associated with HOX Gene Regulation

• No Hox transcripts were found in this study

• However, genes associated with Hox activity were found

• DSP-1 is associated with proteins which form complexes that regulate Hox genes during development (Burns et al., 2011)

• Clone identified to be similar to CREB was highly expressed in 50% DI stages; CREB has been shown to activate Hox genes (Burns et al., 2011)

Discussion of Differentially Expressed Genes Associated with Neuronal Development

• SOX-1 is involved in neuronal determination

• Expression prevents stem cells from differentiating into neurons by blocking the activity of proneural basic helix loop proteins (Burns et al., 2011)

From Figure 4. comparison of microarray results to Q-PCR results (Burns et al., 2011)

Discussion of Gene Expression of Bone Morphogenic Protein-1

• TGF-Beta previously thought to be highly involved in regeneration

• Only member of TGF-Beta superfamily was bone morphogenic protein 1

• Significantly down regulated in blastema and 50% DI stages and increased expression in 95% DI stage

From Figure 4. comparison of microarray results to Q-PCR results (Burns et al., 2011)

What We Learned vs. What We Still Need To Know:

• Study provides insight on molecular basis of regeneration events in Amphiura filiformis

• No Hox genes found in this study• What about DSP-1?• Errors?• Unknown functions of…

• tRNA selenocysteine protein 1• Hyaline

• Involved in developmental morphogenesishttps://media.licdn.com/mpr/mpr/shrinknp_400_400/AAEAAQAAAAAAAAZuAAAAJDdiZTQ4NzU0LWUwOTMtNGY5OC05MzBmLWRkOGIyOTNlOGI4Mg.jpg

The End