Myelination during fracture healing in vivo in myelin protein zero (p0) transgenic medaka line

Highlights

• A mpz (P0)-EGFP transgenic medaka line was produced for tracing myelinating cells.

• mpz-positive cells were found accumulated on the axon at the proximal site of bone fractures.

• mpz-positive cells showed elongation along with the nerve fiber following fracture occurrence.

Abstract

During the fracture healing process, osteoblasts and osteoclasts, as well as the nervous system are known to play important roles for signaling in the body. Glia cells contribute to the healing process by myelination, which can increase the speed of signals transmitted between neurons. However, the behavior of myelinating cells at a fracture site remains unclear. We developed a myelin protein zero (mpz)-EGFP transgenic medaka line for tracing myelinating cells. Mpz-enhanced green fluorescence protein (EGFP)-positive (mpz⁺) cells are driven by the 2.9-kb promoter of the medaka mpz gene, which is distributed throughout the nervous system, such as the brain, spinal cord, lateral line, and peripheral nerves. In the caudal fin region, mpz⁺ cells were found localized parallel with the fin ray (bone) in the adult stage. mpz⁺ cells were not distributed with fli-DsRed positive (fli⁺) blood vessels, but with some nerve fibers, and were dyed with the anti-acetylated tubulin antibody.

We then fractured one side of the caudal lepidotrichia in a caudal fin of mpz-EGFP medaka and found a unique phenomenon, in that mpz⁺ cells were accumulated at 1 bone away from the fracture site. This mpz⁺ cell accumulation phenomenon started from 4 days after fracture of the proximal bone. Thereafter, mpz⁺ cells became elongated from the proximal bone to the distal bone and finally showed a crosslink connection crossing the fracture site to the distal bone at 28 days after fracture.

Finally, the effects of rapamycin, known as a mTOR inhibitor, on myelination was examined. Rapamycin treatment of mpz-EGFP/osterix-DsRed double transgenic medaka inhibited not only the crosslink connection of mpz⁺ cells but also osterix⁺ osteoblast accumulation at the fracture site, accompanied with a fracture healing defect. These findings indicated that mTOR signaling plays important roles in bone formation and neural networking during fracture healing. Taken together, the present results are the first to show the dynamics of myelinating cells in vivo.

Introduction

A fracture, one of the most common traumatic injuries occurring in animals, can be caused by a high force impact or stress, and is well known to be accompanied by severe pain and decline in function. Developmental progression of fracture healing has been studied at the tissue, cellular, and molecular levels [1]. Bone is a complex organ with a capacity for regeneration, though the fracture healing process remains not fully understood. A bone fracture is recognized by the host based on signals from sensory and sympathetic nerves, which are myelinated and located in the periosteum [2]. Previous studies have shown that sensory nerve fibers innervating the periosteum express mechanosensitive C and A-delta nociceptors, and that nerves play an important role in the process of healing of a bone fracture [3,4]. Within minutes to hours following occurrence, a host of neurotransmitters, cytokines, and nerve growth factor are released from cells at the fracture site, which then stimulate, sensitize, and induce ectopic nerve sprouting of sensory and sympathetic nerve fibers, resulting in sharp pain with movement and a dull aching type of pain at rest [3]. However, the role of nerves at the fracture site remains unclear. In this present study, we focused on the nervous system especially myelinating cells, which are known to be involved in coordination of nerve repair [5]. Those cells are thought to convert to a reactive state, and become active for de-differentiation and proliferation when an injury occurs [6].

A large body of bone fracture research has been performed with clinical human and mouse models. However, those systems lack capability to observe the complexity of cell interactions at the fracture site in vivo. In this regard, zebrafish have been identified as a useful model, in that the body is skeletal and in vivo spatiotemporal observations can be performed. Morphogenetic cellular movements and the genetic program that drive myelination are conserved between zebrafish and mammals, thus several studies of myelination have been performed with zebrafish, including larvae and developing stage fish, and also a nerve damage model [[7], [8], [9]]. A major structural protein in myelin known as myelin protein zero (mpz, p0) has received focus and a transgenic mpz promotor study protocol, designed to drive enhanced green fluorescence protein (EGFP), has been created [9].

Medaka, another type of fish, is also known as an effective spatiotemporal in vivo model and has been utilized for bone studies. We have used medaka to study the behavior of osteoblasts and osteoclasts, as well as effects of gravity on bone, as bone dynamism could be visualized [[10], [11], [12]]. Furthermore, a fracture model has been established using the medaka caudal fin, as a specific bone can be fractured without damaging blood vessels, which provides important information that is difficult to obtain with a mouse model [[13], [14], [15]].

For the present study, we established a novel medaka line using the promotor of mpz, to drive expression of EGFP specifically in myelinating glia. We observed mpz-EGFP signals at various ages from embryo to adult stage, and found positive signals starting from the hind brain and developing in the spinal cord, lateral lines, and to the peripheral nerves. Important findings included observation of mpz-EGFP signals in lepidotrichia at 28 days post-fertilization (dpf), which continued to grow and remained clearly visible, owing to the anatomically thin structure of the medaka caudal fin. Also using an established fracture experimental model made for mpz-EGFP medaka [[13], [14], [15]] we performed a systematic fracture experiment with the caudal fin to observe the dynamics of mpz⁺ cells at the fracture site. Those results showed that mpz⁺ cells accumulated at the proximal fracture site for a period of 1 week and showed response to the fracture healing process. Furthermore, we found that neogenetic mpz⁺ cells appeared in specific nerve fibers during the fracture healing process, a phenomenon that was inhibited by an mTOR inhibitor. These present findings will contribute to resolve the role of myelination during the fracture healing process.