Delayed Developmental Loss of Regeneration in Xenopus laevis tadpoles A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of Master of Science In the department of Biological Sciences of the McMicken College of Arts and Sciences by Justin Y. He B.S. Biology, University of the Pacific Committee: Dr. Daniel Buchholz- Chair Dr. Ed Griff Dr. Josh Benoit March 2021 i Abstract: The prospect of spinal cord regeneration in humans is an exciting medical advance, but one that remains elusive from the complicated cellular and molecular mechanisms that prevent regeneration from happening. Various model organisms that do possess regenerative ability have been studied in hopes of understanding how spinal cord regeneration can be facilitated in humans. Recent studies in non-regenerative mammalian organisms however have uncovered the role of T3 signaling pathways in inhibiting regenerative capacity. These previous studies have shown inhibition of T3 in-vitro and in-vivo in various model organisms has increased the capacity for regeneration even in organisms that typically do not have such an ability. My dissertation provides a broad examination of previous literature exploring the barriers to regeneration in a wide range of model organisms, as well as potential therapeutic targets for inducing regeneration. Here, I also show how inhibition of T3 in X. laevis tadpoles allows for increased functional recovery from spinal cord transection. ii © Copyright by Justin He 2021 All Rights Reserved iii Acknowledgements As I conclude my studies at UC in the midst of the COVID-19 pandemic, thank you to all of my friends, colleagues, and family for their love and support in these hectic times. Thank you to my advisor Dr. Daniel Buchholz for his support in my research and writing, and to my lab members for constructive feedback on my presentations during our lab meetings. As I transition into a career as a K-12 teacher, I would also like to thank Dr. Brent Stoffer, the head of the BIOL 1081L & 1082L lab series, for showing us how to effectively teach the material during my time as a TA and for his guidance on transitioning to the world of virtual teaching during this pandemic. Special thanks to him for also providing me with the pedagogical skills necessary to be an effective science educator. iv Table of Contents Chapter 1 Introduction……………………………………..………………………………...…...…………..2 Evolutionary and Developmental Loss of Regeneration………………………..………………...3 Extrinsic Factors Inhibiting Regeneration: Inflammatory Responses and Myelin…………………………………...……..………………….5 Intrinsic Factors: Neural Signaling Pathways and Crucial Neural Genes………………………...…...……………..8 Summary and Next Steps………………………………...………………………………………11 References……………………………...………………...………………………………………12 Chapter 2 Abstract………………………………...………………...………………………………………17 Introduction…………...…………………………………………………….……………………17 Materials and Methods…………………………………………..………….……………………21 Results…………………………………………..………………….……….……………………25 Discussion………………..………………….……………………………...……………………28 Tables and Figures……………………………...………………………………………………..35 Chapter 3 Where do we target? …………………………………………………………………...………..41 Recent advances in our understanding of neural regeneration……………………...…………………………………………...………..43 Implications of facilitating regeneration?..............................……………………………………44 Concluding Remarks ............………............................………………………………………….45 References Cited…...............………............................………………………………………… 47 v Chapter 1 1 Introduction The capacity for neural regeneration specifically recovery from spinal cord injury varies across the animal kingdom (Alibardi, 2019; Lee-Liu et al., 2013). Basal vertebrates of the animal kingdom such as anamniotes including fish and amphibians possess the ability to recover from neural injury post embryonically unlike amniotes including mammals which only possess regenerative ability in the embryonic stages (Kundi, 2013). Understanding the mechanisms of regenerative abilities and developmental loss of regeneration across the vertebrate groups can answer questions on how neural injuries including spinal cord injury can be cured in non- regenerative organisms. Many studies in various model organisms have been conducted to understand cellular and molecular barriers to regeneration. These studies have suggested potential therapeutic targets for facilitating regeneration, detected cellular functions impeding regeneration, as well as identified key regenerative genes that have been lost throughout the evolutionary history of vertebrates. Peripheral nervous system neurons in mammals (PNS) unlike central nervous system neurons have been found to be capable of recovering from injury (Filbin, 2003; Mietto et al., 2015). Both extrinsic and intrinsic factors have been studied in understanding differential ability to regenerate between CNS and PNS neurons. Extrinsic factors include external neural factors such as glial scar formation or myelin and intrinsic factors include internal neural factors such as cytoskeletal organization after injury or expression of certain transcription factors. It has been previously concluded that the different external environments of CNS and PNS neurons are the main factor in differential regeneration ability (David, S; Aguayo, 1981; Reier et al., 2017). This is based off on previous observations showing how PNS neurons transplanted into a CNS 2 environment are unable to elongate despite their know regenerative capabilities (Aguayo et al., 1981). It has also been shown that CNS myelin is inhibitory to regeneration but not PNS myelin (Yiu & He, 2006). The extrinsic environment of CNS neurons is influenced by certain inhibitory molecules that are not expressed around the PNS (Mietto et al., 2015; Toy & Namgung, 2013). These extrinsic factors of the CNS can be targeted to create a more permissive environment for spinal cord injury recovery (Ferguson & Son, 2011; Kim et al., 2012). Here I discuss key studies across various model organisms that show evolutionary and developmental loss of regeneration, extrinsic and intrinsic factors inhibiting regeneration, as well as potential therapeutic targets to facilitate regeneration. Evolutionary and Developmental Loss of Regeneration In many vertebrates, some degree of regenerative ability in the CNS can be found at the earlier stages of development especially in the embryonic stages as it is the stage where there is much potential for neural stem cells to differentiate into their functional form (Alibardi, 2019). In vertebrates that undergo metamorphosis, however, any traces of regenerative ability in CNS neurons cease to exist as development progresses. Why and how metamorphic changes are associated with loss of regeneration is continuing to be studied but various theories have been formulated discussing how developmental loss of regeneration occurs and why mammals and birds experience this phenomenon. From an evolutionary standpoint, it is believed that the expression capabilities of regenerative genes were lost from the transition from water to land due to the need for more specialized organs both internally and externally to perform complex functions on land (Alibardi, 2019). Various inhibitory mechanisms have evolved to suppress 3 neural plasticity as memory and behavioral functions would be lost from constant remodeling of the nervous system. At what stage of life developmental loss of regeneration occurs varies across vertebrates. In humans, regenerative ability is lost at the onset of birth. Other mammals such as opossums possess the ability to recover from spinal cord injury within only 9-12 days postnatal (Mladinic et al., 2009; Wheaton et al., 2020). In certain amphibians such as Xenopus laevis frogs on the other hand, the loss of regeneration occurs during the transition from tadpole to froglet (Gaete et al., 2012; Helbing et al., 2003). The developmental transition from tadpole to froglet is analogous to the transition from fetus to birth in humans as well as developmental loss of regeneration in humans making X. laevis a valuable model organism to study developmental loss of regeneration (Buchholz, 2015). The difference in regenerative capabilities between X. laevis tadpoles and froglets is also evident from other studies that have shown the differences in regenerative ability between tadpoles and froglets. Through histological studies, a negative correlation between neural regrowth across the lesion and developmental stage of X. laevis tadpoles was shown (Muñoz et al., 2015). Lower developmental staged tadpoles experienced a greater degree of neural regrowth across the lesion site than the more mature tadpoles. At the end of 20 dpt (days post transection) lower staged tadpoles had formed an ependymal canal, a central canal of the spinal cord, across the lesion site (Muñoz et al., 2015). While some cellular material did cross the lesion site of higher staged tadpoles at 20 dpt, an ependymal canal was not formed indicating incomplete spinal cord regeneration (Muñoz et al., 2015). Understanding the process of developmental loss of regeneration in these model organisms and what signaling pathways are involved is of great interest in understanding the barriers to regeneration. 4 Metamorphosis is induced by thyroid hormone (T3) suggesting a role of T3 in inhibiting regeneration. T3 is also required for brain remodeling and developmental transition during birth in mammals including humans. Extensive studies