Investigating EZH2 in Zebrafish Craniofacial Bone Development and Fin Regeneration Mychael Solis-Wheeler Texas Tech University SPUR Intern University of Oregon Stankunas Lab

Path of Development to Regeneration Capable in many species EX: Zebrafish Humans Importance Mechanisms Regenerative/Developmental Therapies Cost Efficiency Gene Regulation 2dpa 5dpa 14dpa In Spiderman film series, zebrafish were mentioned along with the H3K27 histone site in the background of a scene, which highlights the potential regenerative research like this. What’s unique about the zebrafish is that it uses a particular type of regeneration called epimorphic regeneration,, which occurs from gene expression/regulation to allow progenitor cells to develop from mature cells that result back to mature cells to regenerate missing bone structures or limbs. Points: To better understand regeneration/bone repair mechanisms To develop therapies for bone disease/fractures To allow faster and cost effective treatments To expand regenerative medical applications Some facts 1.5 million osteoporotic fracture occur each year US medical cost of osteoporosis and fractures is estimated at $22 billion in 2008 according to the NIH.

Histone Modifying Proteins Impact Gene Regulation Histone Modifying Proteins EX: Acetyltransferase Deacetylase Methyltransferase (Ezh2) Demethylase, etc In epimorphic regeneration of the zebrafish, different genes are expressed through regulation to initiate that process. One of the many ways this occurs is by chromatin formation from histone modifying proteins, which in turn represses gene expression when needed. These histone proteins have N-terminus tails that can be modified. Some of these modifications are associated with gene expression/regulation. One of the modifications of interest is the tri-methylation onto lysine 27 of histone 3 that is associated with gene repression. This is the reason we use the zebrafish model to study if this type of modification is involved within its epimorphic regeneration process.

Generating Ezh2 Null Alleles with Genome Editing Null alleles were generated by CRISPR/Cas9, a genome editing tool for loss of function in ezh2 expression. Overt phenotypes observed previously were lack of swim bladder, craniofacial defects, impaired gut formation. When I entered the lab, I wanted to look into characterizing this mutant phenotype further by looking into the craniofacial bone development of the zebrafish larvae. These mutants were generated from crossings, I doubled stained the zebrafish larvae with Alizaran Red, that stains calcified bones as red, and Alzian Blue, which stains cartilage. I then observed over 200 specimens alone under a microscope to look into the craniofacial features for characterizing the mutant phenotype based off random selection before I genotyped them from PCR and RE Digests, followed by taking most of the pictures you will see today. The Ezh2 mutants that I characterized for craniofacial features were observed to be homozygous recessive. Unfortunately, the mutants we worked with did not make it to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. Thus we can infer that the knowledge gained from studying the developmental side of how Ezh2 repression in the zebrafish larvae can impact and share strong connections to the regeneration process in the adults in that the same of not very similar mechanisms that operate and re-capulate the regeneration process in which mature cells are switched into progenator cells to regenerate bone when needed, such as replacing bone rays found in the fins. Thus, highlighting the importance of studying development in order to understand how regeneration occurs. Methods: Embryos were microinjected with this gene editing molecule to generate a loss of function in ezh2 gene expression and were phenotyped/genotyped.

Ezh2 -/- Exhibit Multiple Overt Phenotypes

Question/Hypothesis Question: How does Ezh2 contribute to gene expression during fin regeneration? Hypothesis: Ezh2 has parallel functions in bone development and regeneration in repressing genes within tissues and allow for de-differentiation in progenitor cells.

Craniofacial Schematic of Opercal (OP) To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. .

Reduced OP in Ezh2 Mutant Day 6 Larvae Ezh2 (+/+) Ezh2 (-/-) To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. .

Ezh2 Directs OP Morphogenesis Day 6 Larvae Ezh2 (+/+) Ezh2 (-/-) To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. .

OP Differences Noticed Starting at Day 5 Day 4 Day 5 Day 6 Ezh2 (+/+) n=46 To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. . Ezh2 (-/-) n=31

Craniofacial Schematic of Branchiostegal Rays (BSR) To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. .

Reduced BSR in Ezh2 Mutant Day 6 Larvae Ezh2 (+/+) Ezh2 (-/-) To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. .

BSR Differences Noticed Starting at Day 5 Day 4 Day 5 Day 6 Ezh2 (+/+) n=46 To further investigate the craniofacial defects, I doubled stained larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the branchiostegal ray (BSR), a certain bone that supports jaw development. When characterizing these phenotypes, I set a very conservative standard in my analysis of these bone structures. Although there were occasional fusion features of the BSR 2 and BSR3 that were uncharacteristic for wildtypes, there is do not diminish the lengths of these structures which maybe in the the background of these specimen. Our purpose for identifying phenotypes was to observe how not only how craniofacial bone development was impacted but also how epimorphic regeneration was impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. . Ezh2 (-/-) n=31

Significant Differences in BSR Length Averages Ezh2 (+/+) Ezh2 (+/-) Ezh2 (-/-) To further investigate the craniofacial defects, I doubled stained 6-day larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the BSR, a certain bone that supports jaw development. Our purpose for identifying phenotypes is to observe how not only how craniofacial bone development is impacted but also how epimorphic regeneration is impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. n=32 BSR-2 Avg Length (um): 47 BSR-3 Avg Length (um): 135 n=74 BSR-2 Avg Length (um): 47 BSR-3 Avg Length (um): 131 n=27 BSR-2 Avg Length (um): 10 BSR-3 Avg Length (um): 65

Significant Differences in BSR Length Analysis p<0.0005 i p<0.0005 i p<0.0005 p<0.0005 To further investigate the craniofacial defects, I doubled stained 6-day larval zebrafish with Alcian Blue, that stains for cartilage, and Alizarin Red, that stains for bone, in order to observe bone development. The mutants do not survive to adulthood. However, what was found within the cranial structures of mutants were malformations of opercal, a certain bone that protects the gills, and the BSR, a certain bone that supports jaw development. Our purpose for identifying phenotypes is to observe how not only how craniofacial bone development is impacted but also how epimorphic regeneration is impacted from the knockout of Ezh2. By observing how Ezh2 bone development gain insight of how Ezh2 potential role in bone regeneration since there is a re-activation of developmental genes. n= 133 n= 133

Conclusion Conclusion: Future Research: Ezh2 has roles in regulating genes controlling bone length and patterning in the BSR and bone shaping in the OP. Future Research: Identify genes misregulated by Ezh2 loss of function from antibody staining and in-situ hybridization. Attempt mutant rescue to adulthood by Ezh2 microinjection during development. Identify any differences in cartilage formation between mutant and WT phenotypes. Preliminary results suggest Ezh2 inhibition may impact regeneration in adult zebrafish.

Poor Regenerative Outgrowth from Ezh2 Inhibitor DMSO EZH2i Since larval mutants did not survive into adulthood we observed effects of an Ezh2 inhibitor injected into adult zebrafish in order how Ezh2 inhibition could impact fin regeneration.

Acknowledgements PI: Dr. Kyrn Stankunas Stankunas Lab Mentor: Gabriel Yette Dr. Scott Stewart Dr. Brynn Akerberg Fern Bosada Alex Akerberg Ben Armstrong Kate Karfilis Astra Henner Thomas Forman Justine Nguyen Dr. Charles Kimmel Kimmel Lab Dr. James Nichols John Dowd Adela Chicas-Cruz Eli Cytrynbaum Rest of Lab Institute of Molecular Biology at the University of Oregon University of Oregon SPUR Director Peter O’Day Coordinator Marilyn Drennan Phoebe Penix Rest of SPUR Funding 5R25HD070817-5 NICHD R25 Summer Research Program National Institute of Health (NIH)

Questions?

Ezh2 Null Alleles Lack A Sac I Restriction Site With the CRISPR/Cas9, 7 nucleotides were edited out of the mutants, which is how we genotyped those mutants due to no Sac I site being cut compared to the full WT and Heterozyous WTs.