Biology Department

Northeastern University

Boston, Massachusetts

BASIC RESEARCH PROJECT

Stem cell fate determination during axolotl retina regeneration

Scientific Summary: Degeneration of the human retina is irreversible, but Mexican axolotl salamanders have an amazing ability to regenerate their retinas even after their complete removal. The aim is to understand the cellular and molecular mechanisms that enable salamander retinal regeneration, with the goal of eventually applying this knowledge to restoring damaged retinas in humans.

The axolotl salamander is famous for its enhanced regenerative abilities, including its
limbs and tail. Yet, before our work six years ago, which was funded by RRF, it was
thought that the axolotl may not be capable of retina regeneration. Over the past several
years, Dr. Monaghan’s lab has developed this system into a modern animal model of natural complete retina regeneration. Over the past two years, a graduate trainee, Nicole Calder has continued our previous work to demonstrate retinal regeneration and identify its cellular source definitively. Research proposed for 2026 will continue this in-depth investigation to identify the cellular and molecular basis of axolotl retina regeneration.

Dr. Monaghan’s original discoveries met skepticism from the community, based on previous literature suggesting that the axolotl retina does not regenerate. Therefore, the lab has set out to very rigorously and definitively prove functional retina regeneration in the axolotl. So far, strong evidence has been demonstrated, which suggests that the axolotl’s retina regenerates primarily from a layer of cells called the retinal pigment epithelium (RPE), the dark, protective layer located behind the retina. These RPE cells appear to transform, similar to stem cells, into the various types of neurons required for vision. This discovery contradicts long-held beliefs and opens new therapeutic possibilities.

Plans for 2026

For 2026, Dr. Monaghan is developing a retinal cell atlas using single-cell RNA sequencing in uninjured and regenerating retinas to understand the similarities and differences
between the axolotl and human retina. His lab will also identify the specific genes that
control this transformation by examining which genes turn on and off during
regeneration. Using this data, Dr. Monaghan can identify potential drug targets for stimulating similar regeneration in human eyes. This work is beyond the budget of this proposal, but the support of RRF has enabled considerable progress to meet this goal through continued efforts in the lab and our collaboration with the Baranov Laboratory at
Schepens Eye Institute. Dr. Monaghan’s ultimate goal is to translate these findings into therapies for human patients. By understanding how the axolotl naturally solves the problem of retinal injury, the lab aims to develop strategies to activate similar regenerative processes in human cells.

Progress in 2025

Through the 2025 grant period, Dr. Monaghan’s lab team initiated the development of innovative genetic tools that enable the tracking of the stem cells of the retina. By inserting permanent fluorescent markers into RPE cells, their journey can be tracked as they transform into retinal neurons during regeneration. Preliminary histological results support the hypothesis that RPE cells indeed give rise to multiple retinal cell types, including the light-sensing photoreceptors and neurons that transmit visual information to the brain.

In 2025, the project proposed to develop a retinal atlas and stem cell fate maps of axolotl retina regeneration and to study the molecular mechanisms driving the process. This was accomplished by achieving two primary goals. Firstly, the team progressed toward creating a “family tree” that illustrates the cells involved in retinal regeneration. This extends previously RRF-funded research, where three stem cell sources were developed following retinectomy and NMDA injury; we will now incorporate optic nerve transection as the final injury model. Secondly, lab members worked ti finalize their study of the cells that facilitate regeneration after excitotoxic retinal injury through single-cell RNA sequencing.

In addition, Dr. Monaghan’s lab will conduct new single-cell RNA-seq analyses to enhance our single-cell atlas, perform spatial RNA imaging to pinpoint crucial cell types, and determine which cells change state during the regeneration process.

Specific Aims: Aim 1 will continue to generate lineage and fate maps of the cells driving retina regeneration by comparing three translationally relevant injury models. Aim 2 will identify the molecular players that lead to cell reprogramming and the recruitment of stem cells.

Progress in 2024

The Monaghan research team continues to work on developing the axolotl retina regeneration model to gain insights into the molecular and cellular mechanisms behind
retinal regeneration. Over the past year, the lab created the first cell-type atlas of the axolotl retina and examined its response to NMDA injury, the results of which were presented at the annual ARVO meeting. This research has become the primary focus for Nicole Calder, a second year PhD student, and Emil Kriukov, a visiting MD bioinformatics researcher. The team also conducted a transcriptomic comparison of extracellular matrix genes expressed in the axolotl versus pig retina, which they plan to submit by the new year.

Progress in 2023

Dr. Monaghan’s team continued their research surrounding axolotl retina regeneration to better understand its molecular and cellular processes. Using imaging of transgenic axolotls they generated, the team learned that multiple stem cell sources are involved in regeneration and they identified the cell lineages that contribute to the process. Dr. Monaghan also studied the genes affected during regeneration, particularly in the Notch signaling pathway. This research has led to the graduation of one PhD student, Anastasia Yandulskaya, and the recruitment of a new PhD student, Nicole Calder. Dr. Monaghan and his evolving laboratory team remain committed to continuing to advance our knowledge of axolotl retina regeneration.

This project aims to understand the molecular mechanisms that drive axolotl regeneration of retinal cells.

Throughout 2023, Dr. Monaghan’s laboratory performed imaging of live, genetically modified axolotls that allows the visualization of cell behavior in the regenerating retina. This will be used to identify the cell sources that contribute to the regenerating retina. His team will also compare two different injury models to determine if different cells that mediate regeneration arise from different regions of the eye. Further, they will also compare the molecular signaling between the two injury models to determine if they use common cell signaling to regenerate the retina.

Specific Aims: Aim 1 will perform in-vivo imaging of regenerating retinas in the axolotl. Aim 2 will test whether specific cell signaling pathways identified in our 2022 funding cycle are required for retina regeneration.

Progress in 2022

Analysis of Notch Signaling-mediated Cell Fate determination during Regeneration of the Neural Retina

Dr. Monaghan’s findings from the 2022 funding period finalize the histological description of retinal regeneration, completed the first transcriptomic analysis and multiplexed imaging of gene expression, and confirmed that the Notch signaling pathway plays a critical role in retinal regeneration. This work has culminated in a manuscript that has undergone one round of review and revision and should be accepted in the upcoming days. Over the past two years, Dr. Monaghan has also begun to identify the cell types activated and the molecular mechanisms driving the overall response.

Dr. Monaghan proposed to refine his understanding of the role of Notch signaling in retinal regeneration.  His laboratory team characterized the activity of specific components of this signaling cascade as a new retina forms and give rise to photoreceptors neurons and other cell types.

Specific Aims: The lab investigated how Notch effector genes Hes1 and Hes5 control regeneration of specific cell types in a regenerating retina, focusing on cells of the regenerative retinal pigment epithelial layer. They also engineered a line of transgenic axolotls that will fluorescently reveal Notch activity in specific cells, allowing greater understanding of the mechanisms by which Notch signaling controls retinal regeneration.

Progress in 2021

Dr. Monaghan’s findings from the 2021 funding period have confirmed that the Notch signaling pathway plays a critical role in retinal regeneration. His laboratory team has shown that Notch controls regeneration of photoreceptor neurons and that two of its effector genes, Hes1 and Hes5, are expressed differentially in a regenerating retina. They have also shown that the axolotl retina likely regenerates from the cells of the retinal pigmented epithelium, identifying for the first time the retinal stem cell population in this animal model.

Progress in 2020

In 2020, Dr. Monaghan developed a technique that reveals cell types in the axolotl retina and their gene activity, which will equip the research team to understand the molecular mechanisms behind regeneration.  The team collected preliminary data suggesting that the Notch signaling pathway may dictate what types of cells regrow in the new retina.  Dr. Monaghan’s research confirmed that the regenerated retina re-establishes its connection with the brain, which is necessary for its functionality.  The research also showed for the first time that the axolotl retina contains a type of glial cell that may serve as stem cells during salamander retinal regeneration.

Results Previously Published: In eyes of newt and axolotl salamanders, the retinal pigment epithelium layer, which is adjacent to the neural retina, provides progenitor cells that rebuild the retina after an injury (Islam et al., 2014; Svistunov and Mitashov 1983). Notch signaling pathway, which facilitates inter-cellular communication and regulates gene expression, is active in the retinal pigment epithelium of the regenerating salamander eye, and its perturbation accelerates regeneration of retinal neurons (Nakamura and Chiba 2007). Notch signaling is also active in the mouse retinal pigment epithelium and can induce cultured mouse retinal cells to adopt some features of retinal photoreceptor neurons (Ha et al., 2017; Osakada et al., 2007). These results suggest that Notch signaling and its target genes may provide the key to unlocking regenerative potential in human retinas.