Dissecting NF1: Novel Techniques for Studying Nervous System Diseases and Their Genetic Pathways

Meet The C-P Lab!

Introduction

What is NF1?

Methodology

Findings and Discussion

Conclusions and Future Study

Acknowledgements

References

NACE Career Readiness Competencies

Meet The C-P Lab!

Mia Angulo

Chemical Engineering & Molecular Biology

Class of 2028

Ronald Henkel

Biology & Pre-Medicine

Class of 2026

Ben Callaway

Cell, Molecular, and Structural Biology

Ph.D Student

Caiden Duskey

Lab Assistant

B.S. Biology

Mark Charlton-Perkins, Ph.D.

Assistant Professor, Department of Biology

Introduction

Neurofibromatosis type 1, or NF1, affects 1 in 3,000 children, causing unregulated cell growth in the nervous system from an early age. NF1 generates two major types of tumors: optic pathway gliomas (OPGs) and plexiform neurofibromas (PNFs). Both can cause pain and disfigurement in patients, and are often impacted by other mutated genes. APC, another important cell cycle regulator, has been shown to be one of these genes, causing a higher proportion of PNFs than NF1 alone.

Through a systematic series of genetic knockdowns and knockouts, we have engineered a population of zebrafish with both the NF1 and APC mutations. We study the tumor load in these animals with two primary methods-- dissection and cryosectioning. Spinal dissections have proven especially useful for studying NF1/APC-associated PNFs. Our novel methods let us take a closer look at how the two genes interact, combining the detail of sectioning with the efficiency of a typical body dissection.

Every detail matters when patients are involved, and we are excited to pursue this understanding-- and one day, a cure.

What is NF1?

Our research primarily focuses on neurofibromatosis type 1, or NF1. NF1 is found in around 1 in every 3,000 people and is common in pediatric patients. When functioning correctly, the NF1 gene suppresses cell growth and keeps the cell cycle in check. When mutated, cell growth pathways are triggered and tumors can form in the nervous system.

NF1 generates two major types of nervous system tumors. Optic pathway gliomas (OPGs) and plexiform neurofibromas (PNFs). OPGs form on the optic nerve, often resulting in bulging eyes or impaired vision. PNFs form in the peripheral nervous system, and are commonly observed in the head and neck area. PNFs can press on nearby nerves and are sometimes visible beneath the skin, causing pain and disfigurement. Both can be found in different amounts and severities in NF1 patients, and are often impacted by other mutated cell growth pathways.

From left to right: an MRI image of optic pathway glioma in a human patient. An MRI image of plexiform neurofibroma in a human patient, with tumors gathered on one side of the neck. A side-by-side comparison of an exposed control zebrafish spine and a heavily tumor-laden NF1 zebrafish spine.

How Do We Study NF1?

Our lab works in collaboration with Cincinnati Children’s and Lurie Children’s Hospitals. Both hospitals collect NF1 patient data, tracking their tumor load and noting what other cell growth mutations they have. These mutations can then be induced in zebrafish, which share our NF1 gene. Using the fish, we are able to study thousands of NF1 cases, identifying symptoms and pathways for future treatments.

One of the primary satellite mutations we study is the APC gene, another tumor suppressor found in both humans and zebrafish. NF1/APC patients develop more tumors than those with NF1 alone, often presenting with both OPGs and PNFs.

PNFs typically form on the DRGs, or dorsal root ganglia, which are nerve clusters that extend from the spine. APC is heavily linked to PNF formation, and finding efficient ways to study the spine of affected animals is key to understanding the mutation.

What's the Point?

NF1 is not the most severe genetic disease out there, but it is extremely common. As we learn more about NF1 and its satellite mutations, a cure becomes more of a possibility.

Methodology

Generating an NF1 Zebrafish Model

The NF1 zebrafish model was created by designing a system that lowers NF1 activity only in glial cells, which are support cells in the nervous system. A CRISPR interference system was selected to repress NF1a and NF1b function. The GFAP promoter ensures that the knockdown only occurs in the glial cells, as a full-body NF1 knockout is lethal. Once established, the GFAP::NF1 knockdown line was crossed with an APC knockout line to create an APC heterozygous mutant in the NF1 background.

Methods

We study tumor load in two ways; dissection and sectioning.

Dissection provides a 3-dimensional visual of the animal. Both PNFs and OPGs can be visualized with relative speed, and the dissected fish can be imaged and/or sectioned if needed.

A step-by-step depiction of a body dissection. The fish is first shown prior to dissection. It is then shown opened with organs removed. The final image is a completely clean body with the spine visible.

Images above show measurements of fixed animals that are used in histology or whole mount dissection preparations.

Images of dorsal root ganglia sections in a control, NF1, and NF1/APC fish. Cell nuclei are stained in blue. The NF1 and NF1/APC sections have significantly more of these nuclei, showing excess cell growth.

Histology sections through DRGs are a useful way to capture pathology of hyperplasia in the NF1 and NF1::APC backgrounds (Shown Above). However, we sought to find a way to visualize the gross structure of DRGs and PNF. Spinal dissections are how we meet in the middle.

Spinal dissections are a continuation of body dissections and involve fixation and decalcification. Once the body is sufficiently dissected, excess tissue around the spine and DRGs are removed.

Findings and Discussion

Spinal Dissections.

In the central nervous system, optic nerve thickening has been observed in NF1 and NF1::APC animals, with many having optic pathway glioma. We conducted spinal dissections to observe if NF1 animals have similar thickening in the peripheral nervous system. Spinal dissections reveal that NF1 spines consistently had excess fat deposits surrounding the vertebrae and DRGs. The DRGs are also noticeably wider on NF1 animals, a sign of uncontrolled cell division, the precursor to the formation of plexiform neurofibromas.

Images of a control spine dissection (top) and an NF1 spine dissection (bottom.) The NF1 spine is much less clean, with thickened nerves and excess fat deposits surrounding the vertebrae.

Due to decalcification of the vertebral column, these bones can be removed to visualize the spinal cord, along with afferent and efferent nerves and dorsal root ganglia (DRG). A small loss of nerve tissue occasionally occurs during the process, but mostly intact nerves and DRGs are observed. DRGs have the potential to be conserved, as shown in red below.

Three images of a control zebrafish spine with the vertebrae partially removed. Thin nerves extend from the spinal cord, and are circled and identified as DRGs.

Conclusions and Future Study

Conclusions

Spinal dissections are a viable method for observing NF1 and its modifiers' effect on the peripheral nervous system. Notably, dissection provides a way to grossly visualize DRGs and compare them directly to those of a healthy animal. Our novel methods have demonstrated that there are notable differences between NF1 or NF1/APC spines and healthy spines, including excess fat deposits and nerve thickening.

Our findings also support that the NF1 phenotypes in our animal model is similar to pathologies for NF1 in humans, and thus, extremely valuable for studying this disease.

Future Work

We plan to continue to refine our spinal dissection technique in NF1 and NF1/APC animals. DRG exposure without nerve tissue loss would have incredible value for imaging and study, allowing us to understand NF1 in even greater depth. We also plan to continue the study of APC and other modifiers through dissection, sectioning, and physical observation of the animals.

There is no cure for NF1, and current therapies are highly refractory. Using the zebrafish model to study modifiers will allow for patient-specific therapies to be developed in the future.

The following is an image of the poster presented at the 2026 Undergraduate Research Forum

Acknowledgements

Research

Dr. Carlos Prada (Lurie Children’s Hospital)

Dr. Nancy Ratner (Cincinnati Children’s Hospital)

C-P Lab (Miami University Department of Biology)

C-P Lab Members:

Mark Charlton-Perkins

Caiden Duskey

Ben Callaway

Joydip Barua

Anna Statsenko

Itunu Adewoye

Nelchi Prashali

Ronald Henkel

Lucy Bailey

Dylan Ball

Michelle Townsend

Sarah Alhilu

John Kugler

Sanee Scavo

Mia Angulo

Funding

NINDS: 1R01EY038719-01

Miami University

References

1.Kaufman, L. M. & Doroftei, O. Optic glioma warranting treatment in children. Eye 20, 1149–1164 (2006).

2.Grover, S. B., Kundra, R., Grover, H., Gupta, V. & Gupta, R. Imaging diagnosis of plexiform neurofibroma-unravelling the confounding features: a report of two cases. Radiol. Case Rep. 16, 2824–2833 (2021).

3.Gutmann, D. H., Parada, L. F., Silva, A. J. & Ratner, N. Neurofibromatosis type 1: modeling CNS dysfunction. J. Neurosci. 32, 14087–14093 (2012).

All other images and illustrations were produced by members of the Charlton-Perkins Research Laboratory.

NACE Career Readiness Competencies

Technology

We use cutting-edge digital and biological techniques on the daily. Genetic engineering is a constantly changing field, and we pride ourselves on staying up to date with our methods and use of technology.

Teamwork

Nothing in our lab could be accomplished alone. We have a strong team of undergraduate, graduate, and professional researchers who dedicate themselves to their work-- and one another-- without fail.

Career Development

Biotechnology has exploded in recent years, and the skills we build in this lab translate directly to the industry. Each of our members are working towards impactful careers, including medicine, professional research, and the gene therapy industry.

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