Hyde Lab: Ryan Thummel

Assistant Professor

Department of Anatomy and Cell Biology
Department of Ophthalmology
Wayne State University School of Medicine
540 E. Canfield Ave.
Detroit, MI 48201
Phone: (313) 577-7762
Email: rthummel@med.wayne.edu

1998	University of Notre Dame Notre Dame, Indiana	B.A. Psychology (Cum Laude)
2004	University of Kansas Medical Center Kansas City, Kansas	Ph.D. with Honors, Molecular and Integrative Physiology
	Thesis Title: Genetic analysis of Hoxc13 orthologs in mice and zebrafish
	under Alan Godwin
Present	University of Notre Dame, Center for Zebrafish Research	Postdoctoral Research Fellow
	Project: Molecular Genetic Analysis of Zebrafish Retinal Regeneration
	under David R. Hyde

Many devastating forms of inherited eye diseases result in irreversible blindness because humans lack the ability to regenerate their dying or diseased rod and cone photoreceptors. My work focuses on understanding why some species, like zebrafish, maintain the ability to regenerate their retinal neurons following damage.

Prior to my joining the lab, a large multi-timepoint microarray experiment was performed during intense light-induced zebrafish retinal regeneration (Kassen, et al., 2006). This generated a list of genes that are increased in expression during regeneration of zebrafish rods and cones. My primary project was to develop an in vivo technique to reduce the expression from these genes and test their role during retinal regeneration. I accomplished this by electroporating anti-sense morpholino oligonucleotides into the adult zebrafish retina. Morpholinos are synthetic oligonucleotides designed to bind to the 5’ untranslated region of the mRNA of a gene of interest. Once bound, the morpholino prevents translation by sterically hindering the loading or progression of the translation machinery. Thus, the level of the protein of interest is “knocked-down” the morpholino. By combining the microarray data set with the in vivo knockdown technique, I ultimately hope to find genes that are candidates for inducing regeneration of damaged human retinas.

The first gene selected for protein knockdown was PCNA (proliferating cell nuclear antigen). PCNA is highly up-regulated during retinal regeneration by a specialized glial cell in the retina, termed Müller glia. Müller glia cells divide at 36 hours of constant light exposure (Figure 1), and are the source of the neuronal progenitors during retinal regeneration of rods and cones. As PCNA is needed for cell division, I tested the necessity of Müller glia proliferation on the regeneration of rods and cones by knocking down PCNA levels during retinal regeneration. This was accomplished by making a small incision into the cornea and injecting a morpholino targeted against PCNA into the vitreal space of the eye (Figure 2). Next, the morpholino was driven into the retinal cells with an electroporation event across the eye. Finally, the fish underwent damage of the rod and cones with intense light exposure. I harvested eyes at various timepoints and analyzed them for PCNA knockdown and rod and cone photoreceptor regeneration. To control for non-specific effects of the electroporation I used a standard control morpholino, which has no target in the zebrafish genome. I determined that a morpholino targeted against the pcna gene knocked-down PCNA protein levels in zebrafish embryos and that injection and in vivo electroporation of the pcna morpholino during intense light exposure inhibited Müller glial cell proliferation (Figure 3). PCNA knockdown resulted in specific effects on Müller glia, including decreased expression of the glutamine synthetase enzyme, loss of Müller glial processes, and Müller glial cell death, while amacrine and ganglion cells were unaffected by reduced PCNA levels. Finally, using histological and immunological methods, we showed that long-term effects of PCNA knockdown resulted in the failure to regenerate rod photoreceptors and a disorganization of the regenerated cone photoreceptors (Figure 4). These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with recent data demonstrating Müller glia likely function as neuronal stem cells in regenerating teleost retinas. This work is currently being prepared for publication.

I have three additional projects which reflect my varied interests in development and regeneration, including the analysis of the retinal development mutant termed platinum, utilizing transgenic lines to mark progenitor cells in zebrafish fin and retinal regeneration, and analyzing the early signaling events following retinal damage in the adult zebrafish.

Figure 1. Localization of PCNA in the Tg(gfap:EGFP)^nt albino zebrafish retina during light treatment. EGFP expression in the Müller glial cells in the Tg(GFAP:EGFP)^nt albino zebrafish is examined relative to PCNA immunolocalization during the intense light treatment. At 0 hrs, PCNA expression is occasionally detected in a single cell of the ONL, which likely corresponds to a rod precursor cell. At 36 hrs, (panel B), increased PCNA expression is detected in the INL. This PCNA expression colocalizes with the EGFP expression in the Müller glial cells. At 72 hrs (panel C), the nuclei of EGFP-expressing Müller glial cells do not colabel with PCNA. However, PCNA expression is localized to neuronal progenitors, which have begun to migrate from the INL to the ONL. This migration persists through 96 hrs (panel D). These results suggest the initial Müller cell proliferation event, which occurs subsequent to the light-induced photoreceptor damage, produces a population of neuronal progenitor cells. These newly generated progenitors continue to proliferate, while proliferation of the Müller glial cells ceases.

Figure 2. Injection and electroporation of morpholinos into the adult zebrafish retina. Morpholino injection and electroporation was performed immediately prior to light treatment. Dark-treated adult albino zebrafish were anesthetized in 2-phenoxyethanol and a small incision was made in the cornea just adjacent to the iris using a sapphire blade scalpel (panel 1). A Hamilton syringe was used to inject 0.5 ml of 3.0 mM morpholino solution into the vitreous of the left eye, while the right eyes were either injected with only buffered saline not injected at all (panel 2). Electroporation of the left eye was performed using a CUY21 Square Wave Electroporator (Protech International, Inc., San Antonio, TX) immediately following injection (panel 3). A 3 mm diameter platinum plate electrode was used to direct the morpholino (which has a slightly positive charge due to the lissamine tag) to the dorsal half of the eye. The fish were then revived and placed directly into the light treatment. Following various days of light treatment (or post-light treatment) eyes were harvested for analysis.

Figure 3. Injection and in vivo electroporation of anti-PCNA morpholino during constant light exposure knocksdown PCNA. Dark-adapted adult albino zebrafish were injected and electroporated with either the Standard control morpholino (S.C. MO) or a PCNA morpholino (PCNA MO) and exposed to constant light. Retinas were harvested after 1, 2, and 3 days in constant light and immunolabeled with anti-PCNA (shown in green) and anti-Rhodopsin (shown in blue). In the control retinas (A, C, and E), light exposure results in a progressive accumulation of PCNA-positive cells within the INL and ONL (arrows). In contrast, the PCNA morpholino-injected retinas lack any PCNA-positive cells (Panels B, D and F).

Figure 4. Long-term effects of PCNA knockdown include rod and photoreceptor loss and disorganization. A. At 28 days following light treatment, histological sections of un-injected retinas show a regenerated outer nuclear layer (ONL), cone cell layer (CC) and rod outer segments (ROS). Rhodopsin staining (panel C) reveals regenerated rod outer segments. B. At 28 days post light treatment, histological sections from PCNA morphant retinas show an absence of a distinguishable rod outer segment layer, very few nuclei present in the ONL (arrow), and large sections absent of long single cones (asterisk). Rhodopsin immunostaining confirms the absence of mature rod outer segments (panel D), indicating that rod photoreceptors do not regenerate without Müller glia proliferation. Immunolocalization of blue opsin shows the regeneration of long single cones in the un-injected retinas (panel E), but large areas absent of long single cones in PCNA morphant retinas (panel F). In contrast, zpr-1 staining shows the presence of double cones in both un-injected (panel G) and PCNA morphant retinas (panel H). However, the double cones present in the PCNA morphant retina are severely disorganized and truncated (panel H).

Refereed Manuscripts

1. R. Thummel, L. Li, C. Tanase, M. P. Sarras, Jr., and A. R. Godwin. Differences in Expression Pattern and Function between Zebrafish hoxc13 Orthologs: Recruitment of Hoxc13b into an Early Embryonic Role. Developmental Biology (2004) 274 (2): 318-333.

2. R. Thummel, C. T. Burket, J. Brewer, M. P. Sarras, Jr., L. Li, M. Perry, J. McDermott, B. Sauer, D. R. Hyde, and A. R. Godwin. Cre-mediated Site-specific Recombination in Zebrafish Embryos. Developmental Dynamics (2005) 233 (4):1366-77.

3. S. Bai, R. Thummel, A. R. Godwin, H. Nagase, Y. Itoh, L. Li, R. Evans, J. McDermott, M. Seiki, and M. P. Sarras, Jr. Matrix metalloproteinase expression and function during fin regeneration in zebrafish: analysis of MT1-MMP, MMP2 and TIMP2. Matrix Biology (2005) 24 (4):247-60.

4. R. Thummel, S. Bai, M. P. Sarras Jr., P. Song, J. McDermott, J. Brewer, M. Perry, X. Zhang, D. R. Hyde, A. R. Godwin. Inhibition of Zebrafish Fin Regeneration Using in vivo Electroporation of Morpholinos Against fgfr1 and msxb. Developmental Dynamics (2006) 235: 336-346.

5. R. Thummel, C. T. Burket, and D. R. Hyde. Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration. ScientificWorld Journal. 2006 Dec 15;6:65-81.

6. R. Thummel, M. Ju, L. Li, M. P. Sarras, Jr., and A. R. Godwin. Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration. Dev Genes Evol (2007) Apr 17. To link directly to journal and full article, click here.

7. Thummel, R., Kassen, S.C., Montgomery, J.E., Enright, J.M., Hyde, D.R. (2008) Inhibition of Müller glial cell division blocks regenration of the light-damaged zebrafish retina. Dev Neurobiol. 2008 Feb 15;68(3):392-408.

8. Yin, V.P., Thomson, J.M., Thummel, R., Hyde, D.R., Hammond, S.M., Poss, K.D. (2008) Fgf-dependent depletion of microRNA-133 promotes appendage regeneration in zebrafish. Genes Dev. 2008 Mar 15;22(6):700-5.

9. Burket, C.T., Montgomery, J.E., Thummel, R., Kassen, S.C., LaFave, M.C., Langenau, D.M., Zon, L.I., Hyde, D.R. (2008). Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters. Transgenic Res. 2008 Apr:17(2):265-79. Epub 2007 Oct 30.

10. Hoptak-Solga, A.D., Nielsen, S., Jain, I., Thummel, R., Hyde, D.R., Iovine, M.K. (2008) Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration. Dev Biol. 2008 May 15;317(2):541-8. Epub 2008 Mar 12.

11. Kassen, S.C., Thummel, R. Burket, C.T., Campchiaro, L.A., Harding, M.J., and Hyde, D.R. (2008). The Tg(cyclin B1:EGFP) transgenic zebrafish line labels proliferating cells during retinal development and regeneration. Mol. Vis 14:950-962.

12. Thummel R., Kassen S.C., Enright, J.M., Nelson, C.M., Montgomery, J. E., and Hyde, D.R. (2008). Characterization of Muller glia and neuronal progenitors in zebrafish adult retinal regeneration. Exp. Eye Res. 87: 433-444.

13. Kassen, S.C., Thummel, R. Campochiara, L.A., Harding, M.J., Bennet, N.A., and Hyde, D.R. (2009). CNTF induces photoreceptor neuroprotection and Muller glial cell proliferation through two different signaling pathways in the adult zebrafish retina. Exp. Eye Res. 88:1051-1064.

14. Craig, S.E.L., Thummel, R., Ahmed, H., Vasta, G.R., Hyde, D.R., and Hitchcock, P.F. (2010). The zebrafish galectin Drgal1-L2 is expressed by proliferating Müller glia and photoreceptor progenitors and regulates the regeneration of rod photoreceptors. Invest Opthalmol Vis Sci. 2010 Jan 13 (Epub ahead of print)