Regenerating axolotl retinas regrow diverse cell types with modulation by Notch signaling and reconnect to the brain

Yandulskaya, Anastasia S.; Miller, Melissa N.; Ansaripour, Ronak; Carrier, Rebecca L.; Monaghan, James R.

doi:10.1101/2022.04.28.489898

Cited by 2 publications

(2 citation statements)

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“…Validation and Confirmation: Immunofluorescence serves as a valuable tool to validate the presence of ion channels at the protein level, confirming the findings from RNA sequencing. FISH, on the other hand, corroborates the RNA expression patterns from single nuclear sequencing data, reinforcing the reliability of transcriptomic data 163 .…”

Section: Complementary Nature Of Techniquessupporting

confidence: 63%

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Cartilage mechanobiology during limb growth

Kondiboyina

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The mechanobiology of cartilage during limb growth represents a complex interplay between mechanical forces and biological processes. However, the fundamental processes that involve the formation of cartilage from mesenchymal stem cells are not fully understood. Further, the cellular level response of cartilage in its native environment under physiological load is not fully characterized. This thesis aims to bridge critical gaps in our understanding of limb development and regeneration by investigating the nuanced interactions between mechanics and cellular processes within cartilaginous tissues.Identifying these research gaps, this thesis encompasses three specific aims. Aim 1 focuses on characterizing the viscoelastic material properties of growing limbs, employing axolotls as an animal model. Our results reveal significant increases in both instantaneous and equilibrium shear moduli during limb regeneration, coupled with notable changes in short-and long-term stress relaxation times. The glycosaminoglycan content also increases during development. Aim 2 explores the calcium signaling response of in-situ chondrocytes under physiologically relevant cyclic loads and dynamic hydrostatic pressure. Our findings underscore a strain rate-dependent increase in the percentage of responsive cells under compressive loads, with non-distinct time characteristics across loading conditions. Conversely, low magnitude dynamic hydrostatic pressure showed no significant impact on calcium signaling in chondrocytes. Aim 3 investigates the expression of mechanosensitive ion channels (TRPV4, PIEZO1, and PIEZO2) during axolotl limb regeneration. The study unveils the presence of TRPV4 and PIEZO2 in blastemal cells during early and late regeneration, with heightened expression in the condensing mesenchyme during late regeneration.These findings taken together shed light on the dynamic changes in the mechanical environment during limb growth and the intricate interplay between mechanics and cellular responses. The implications of abnormal mechanobiological processes are profound, contributing to developmental disorders and musculoskeletal diseases. Understanding these fundamental mechanisms under physiological conditions opens avenues for therapeutic strategies aimed at promoting proper limb development and mitigating skeletal abnormalities. Future research will focus on elucidating the functional roles of mechanosensitive channels during regeneration and further expanding our understanding of the complex interconnections between mechanics and skeletal biology. iii I am deeply grateful to my colleagues at the Shefelbine lab, past, and present, whose support and camaraderie have been invaluable. Quentin Meslier, Soha Ben Tahar and Lindsey Young, thank you. Special acknowledgment goes to Noah Mooney for his remarkable work on engineering the hydrostatic pressure system. Working alongside him was truly enjoyable. iv Special thanks to Dr. Helen Markewich for training me in essential laboratory skills. I extend my appreciation to the support...

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Section: Complementary Nature Of Techniquessupporting

confidence: 63%

“…The sections were then stained with DAPI nuclear stain (1:5000), washed with PBS, followed by DEPC water, and finally, mounted in Slow-fade™ (Invitrogen) with 1.5 microscope cover glass (Fisher Scientific). To maintain RNA integrity within the tissue throughout the procedure, we consistently used RNAse-away spray 161,163 .…”

Section: Mrna Staining and Imagingmentioning

confidence: 99%