1. Cell signaling in development and regeneration of the zebrafish (Danio rerio) retina
Jenny Lenkowski, PhD
Goucher College
Department of Biological Sciences
BCPS Professional Study Day
August 18, 2016
I received my bachelor’s degree from Brown University and my PhD at Tufts University. I then worked in a research lab at the University of Michigan prior to joining the faculty at Goucher College two years ago. I was drawn to the college because it supports both teaching and research in a small liberal arts college environment. I will talk about my research at Goucher that focuses on how the retina (1) develops and (2) regenerates in zebrafish, a commonly used research vertebrate that is also found in pet stores. (1) The retina is part of the central nervous system, so my research is about how neurons and glia in the nervous system develop and how cell communication regulates that development. (2) Zebrafish are also capable of regenerating many damaged or destroyed tissues, including the heart, fin, lateral line, and neurons in the central nervous system. To explore this in zebrafish, my students and I kill the neurons in the retina with light that is comparable to looking at the sun for 30 minutes, and then we can examine how retina-specific adult stem cells regenerate the neurons. Humans have the same cell type in our retinas, but rather than acting like a stem cell to regenerate lost neurons, they make a scar when similar damage occurs. I will also touch on previous research that I am considering revisiting that examined how exposure to a common weed killer affect animal development and ways in which these research topics may be explored in a classroom.
What courses do you teach so I can try to keep that in mind?

2. Zebrafish 1 cm 0.5 mm www.ceb.utk.edu/zebraC60.html
McGee et al, EHP, 2012

3. The Vertebrate Eye MS-LS1-3 Barbosa-Sabenero et al, 2012
MS-LS4-2.Apply scientific ideas to construct an explanation for the anatomical similarities and differences among modern organisms and between modern and fossil organisms to infer evolutionary relationships.
Barbosa-Sabenero et al, 2012

4. The Retina Choroid Retinal Pigment Epithelium Outer nuclear layer
MS-LS1-3
The Retina
Choroid
Retinal Pigment Epithelium
Outer nuclear layer
Cone & Rod Photoreceptors
Inner nuclear layer
Interneurons
Müller glia
Ganglion cell layer
Optic nerve
Barbosa-Sabenero et al, 2012

5. Retinal development Eye cup morphogenesis & differentiation
Optic vesicle
2.1 ~ 3.5 days
Optic vesicle reaches ectoderm and forms an invagination to form the eye cup
Video found here too:
Optic cup
Hitchcock & Raymond, 2004

6. (eye development/regeneration)
TGFβ cell signaling pathway regulates proliferation and differentiation during retinal development and regeneration in zebrafish.
Ligands
Receptor IIb
Receptor Ib P
Tgif1 P
Smad
When the Activin/Nodal/TGFβ ligand binds to the Type II receptor, the receptor recruits and phosphorylates the Type I receptor. The Type I receptor phosphorylates the effector Smad 2 or Smad 3, which then binds to the co-Smad4. The Smad complex translocates to the nucleus to affect downstream target genes including ascl1a. Tgif1 and Six3 are known transcriptional repressors of Smad2/3-mediated TGFβ signaling.
Furthermore, during rat retinal development, TGFβ signaling increases when progenitor proliferation decreases and inhibiting TGFβ signaling increases proliferation of Müller glia in vivo (17). In addition, in the first 10 postnatal days, TGFβ signaling is neuroprotective in the retina of mice (20
Controls the amount of neural proliferation in the retina
During mammalian retinal development,
Increased activity of TGFβ pathway increase in differentiation.
Pathway is inhibited  Müller glia proliferation continues (Lenkowski et al., 2013)
Mature retinal cells in mammals exert control over the Müller glia via the TGFβ pathway (Close et al. 2005)
TGFβ pathway involved in slowing retinal cell proliferation during development as well as regeneration after injury (Close et al. 2005) (Jazwinska et al. 2007)
Six3b
Co-Smad
PROLIFERATION
(eye development/regeneration)

7. Manipulating TGFβ signaling
Use a chemical to block TGFβ signaling
Use fish with mutations in endogenous inhibitors
(Inhibit a pathway that inhibits proliferation in the retina = upregulate the pathway)
Regulation of TGFβ signaling has a different role in the tail fin than in the retina.
Tail fin: TGFβ signaling required to maintain the proliferative potential of the blastema
Retina: inhibition of TGFβ signaling necessary for development and regeneration to occur
(Jazwinska et al. 2007)

8. Methods Expose wild type embryos to chemical inhibitor
 collect embryos  fluorescent immunohistochemistry
Look at cell proliferation with antibodies
Mention which cells we were going to look at (zpr1, pcna, gFAP)
Different proteins we wanted to look at
Started with PCNA, plan to do GFAP and zpr1

9. Manipulating TGFβ signaling & observing at 3 dpf
Still working out issues, been through experiment using inhibitors so process is being mastered
Differential interfering contrast
PCNA in red
Put in length of development (2 dpf)

10. Regeneration in Multi-cellular Organisms
MS-LS1-5
MS-LS4-2
HS-LS1-4
Abcam
Novartis
Blogs.nature.com
Carolina Biol.
Carolina Biol.
marietta.edu/~biol/biomes/wetlands.htm
MS-LS4-2.Apply scientific ideas to construct an explanation for the anatomical similarities and differences among modern organisms and between modern and fossil organisms to infer evolutionary relationships.
HS-LS1-4.Use a model to illustrate the role of cellular division (mitosis) and differentiation in producing and maintaining complex organisms.
Hydra undergo morphollaxis – change of shape without proliferation
In plants – have indeterminant growth. Will regrow leaf or root very similarly to how some animals can regrow limbs. de novo generation of tissue from clippings occurs when the plant develops a new meristem where the stem cells reside and there is sometimes an intermediate cell mass (callus).
MS-LS1-5 EXPLANATION: Thought that fish may have this capacity, as they also exhibit some extent of indeterminant growth – larger space & enough nutrition = ongoing growth
Compensatory regeneration such as that in the mammalian liver where cells maintain their identity but proliferate to replace mass and function.
Proliferation of differentiated cells
Proliferation of stem cells
Dedifferentiation of mature cells that then generate new cells
Transdifferentiation of mature cells into a different cell type
L. da Vinci

11. Regeneration in Multi-cellular Organisms
Neoblast
Blastema
1 day
7 days
Neoblast = planaria stem cell
blastema- which is a heterogenous mix of progenitor cells
Simple experiment for which you need Deer Park water, “knives” made out of thick plastic, and practice on gelatin “worms”
B. Pearson

12. Regeneration in Multi-cellular Organisms
1 day
Dedifferentiation
Satellite cells
Blastema
21 days
Epimorphic
Limb forms from a blastema
Satellite cells in skeletal muscles are resident stem cells
Cells also dedifferentiate
Need nerves for successful limb regeneration
40 days
colinfarrelly.blogspot.com

13. Regeneration in Multi-cellular Organisms
Before
Amputated
Blastema
10 days
17 days
Humans do regenerate your skin and lining of your intestine. Your hematopoietic system. These are all for maintaining homeostasis though. In response to injury, mammals and humans are extremely lacking when compared to the other animals I have highlighted.
Epimorphic
Wound healing and blastema formation
Putting a skin flap over the wound inhibits a blastema.
The main difference here is that no complex structures are regenerated. You cannot remove a joint and expect the joint to regenerate.
Fernando et al, 2011

14. Regeneration of the Central Nervous System
Fish
Mammal
Brain
Spinal cord
Retina  
Double check
 Catfish taste buds (Olmstead, 1920)
Cranial nerves
 Olfactory nerve    
REGENERATION
SCAR

15. Ongoing neurogenesis in adult vertebrates
Radial glia
Humans have neurogenesis in the sub-germinal zone of the dentate gyrus, the subventricular zone in the lateral ventricle
Lenkowski & Raymond, 2014
Hindawi.com

16. The Retina Fluorescent immunohistochemistry Say that they are cones
Vithelic et al, 2006
Lenkowski

17. Transgenic gfap:eGFP zebrafish (glial fibrillary acidic protein)
GFP is expressed in Müller glia
lens
5 days post-lesion (dpl)
Cones
Bernardos & Raymond 2006; Bernardos et al, 2007

18. Acute light lesion paradigm
Cones - 3 days post lesion
Light lesion kills predominantly the cone photoreceptors.
Mention that the fish get dark after lesioning – you know they’ve been blinded.
Ryan’s study comparing paradigms

19. Retinal neuron regeneration
Cone photoreceptors
Nuclei
Bernardos et al 2007

20. Regenerative response of Müller glia
Approximate timing
0 days
1 day
2 days
3 days
5 days
Photoreceptors
Müller glia
Mammalian Muller glia are stimulated in response to damage, and can divide. Initial damage response can be neuroprotective – buffer potassium, take up glutamate, release growth factors and cytokines. BUT, they can enter a pathological state and release pro-inflammatory cytokines and nitric oxide and generate a glial scar.
Activate
 GFAP
Proliferate
Make a neural progenitor
Redifferentiate
Re-express glial markers
Markers
Dedifferentiate
Re-express stem-cell markers

21. General injury response in Müller glia
stress response, inflammation, gliosis, and cell adhesion/migration
exposure to TGFβ ligands inhibits proliferation of Müller glia, including growth factor-induced proliferation (17-19).
We know that members of the TGFbeta pathway ARE expressed in the retina during regeneration.
Lenkowski & Raymond, 2014

22. Tgif1 Six3 tgif1-/-;six3b-/- =  TGFβ signaling = proliferation
TGFβ-interacting factor In proliferating cells of the retina during mouse development, in proliferating cells during retina regeneration, and in neural stem cells in the brain (Satoh & Watanabe, 2008; Lenkowski et al, 2013) Regulates cell proliferation and differentiation in vertebrate and invertebrate eye development
Six3
Both are upregulated in MG after lesion per Zhao’s microarray
tgif1-/-;six3b-/- =  TGFβ signaling = proliferation

23. Homozygous mutant tgif1-/- fish generate fewer proliferating progenitors
PCNA
Müller glia
Nuclei
Wild-type
tgif1-/-
Lenkowski et al, 2013

24. Mutant fish have regeneration defect
14 days post lesion
Cell junctions (ZO-1)
Nuclei
Wild type
tgif1-/-
tgif1-/-;six3b-/-
Lenkowski et al, 2013

25. Evidence of scarring after lesion in tgif1-/-;six3b-/- retina
Wild-type
tgif1-/-;six3b-/- (=  TGFβ signaling)
Cell junctions
Zonula occludens-1 (ZO-1) immunofluorescence in white to detect tight junctions on whole mount retinas at 14 dpl in wild-type (A) and tgif1-/-;six3b-/- (B) fish retinas. Lesioned area is above the dotted line as determined by the disrupted photoreceptor pattern that is below the line (1, 2). Arrows indicate = dense ZO staining. Scale bars = 50µm.
(50, 52, 53). Furthermore, TGFβ is a known regulator of GFAP expression (54, 55), so one may predict that increased TGFβ signaling could lead to an increased gliotic response in Müller glia. Following acute light lesion, zebrafish Müller glia also upregulate GFAP
). TGFβ signaling induces a reactive gliotic response in the mammalian retina that can result in a glial scar, and inhibiting TGFβ signaling reduces scarring (21, 22).
There is less proliferation and less regeneration in mutant fish
Is a scar forming??
Lenkowski

26. Examining protein components of scars

27. Evidence of scarring after lesion in tgif1-/-;six3b-/- retina
Wild-type
tgif1-/-;six3b-/- (=  TGFβ signaling)
Cell junctions
Zonula occludens-1 (ZO-1) immunofluorescence in white to detect tight junctions on whole mount retinas at 14 dpl in wild-type (A) and tgif1-/-;six3b-/- (B) fish retinas. Lesioned area is above the dotted line as determined by the disrupted photoreceptor pattern that is below the line (1, 2). Arrows indicate = dense ZO staining. Scale bars = 50µm.
(50, 52, 53). Furthermore, TGFβ is a known regulator of GFAP expression (54, 55), so one may predict that increased TGFβ signaling could lead to an increased gliotic response in Müller glia. Following acute light lesion, zebrafish Müller glia also upregulate GFAP
). TGFβ signaling induces a reactive gliotic response in the mammalian retina that can result in a glial scar, and inhibiting TGFβ signaling reduces scarring (21, 22).
There is less proliferation and less regeneration in mutant fish
Where is the cell cycle being disrupted??
Lenkowski

28. Examining the cell cycle in development and regeneration
Fluorescence ubiquitination cell cycle indicator Miyawaki lab modified it for zebrafish

29. Generate a gfap:zFUCCI fish
mCherry-Cdt1 to visualize G1 and mAzamiGreen-Geminin to visualize S/G2/M phases in glial cells
traditional restriction digest and ligation subcloning methods after the modification of one restriction site using annealed oligonucleotide cloning
Will examine tgif1;ascl1 double mutant larvae
In mutant fish, characterize expression of proteins that are highly expressed in glial scars
Examine retina development in tgif1;six3b mutants
Characterize timing of retina development
With zFUCCI
Rescue experiments – expose mutants to inhibitors of TGFβ signaling
With D. Meir-Levi

30. Acknowledgements Nikon Ti-E Inverted Microscope
Goucher College Summer Research Program Maeve Downey Dani Meir-Levi Katie Brandt
Nikon Ti-E Inverted Microscope

31. Questions?

33. Imaging & analysis options in the classroom
The Smart Phone
The Eye Piece Camera
(Dino-Lite)
ImageJ, morphometrics

34. Developmental Biology
Zebrafish embryos with GFP in the central nervous system

35. Evolution of photodetecting organs