Development of type I/II oligodendrocytes regulated by teneurin-4 in the murine spinal cord

In the spinal cord, the axonal tracts with various caliber sizes are myelinated by oligodendrocytes and function as high-velocity ways for motor and sensory nerve signals. In some neurological disorders, such as multiple sclerosis, demyelination of small caliber axons is observed in the spinal cord. While type I/II oligodendrocytes among the four types are known to myelinate small diameter axons, their characteristics including identification of regulating molecules have not been understood yet. Here, we first found that in the wild-type mouse spinal cord, type I/II oligodendrocytes, positive for carbonic anhydrase II (CAII), were located in the corticospinal tract, fasciculus gracilis, and the inside part of ventral funiculus, in which small diameter axons existed. The type I/II oligodendrocytes started to appear between postnatal day (P) 7 and 11. We further analyzed the type I/II oligodendrocytes in the mutant mice, whose small diameter axons were hypomyelinated due to the deficiency of teneurin-4. In the teneurin-4 deficient mice, type I/II oligodendrocytes were significantly reduced, and the onset of the defect was at P11. Our results suggest that CAII-positive type I/II oligodendrocytes myelinate small caliber axons in the spinal cord and teneurin-4 is the responsible molecule for the generation of type I/II oligodendrocytes.

the discovery by del Río Hortega, Butt and his colleagues identified carbonic anhydrase II (CAII) as a marker for type I/II oligodendrocytes 16,17,19 . They demonstrated that CAII-positive cells in the anterior medullary velum (AMV) extended their complexly arborized processes to the small diameter axons. Regarding CAII staining, the remarkable upregulation of CAII in oligodendrocytes in demyelinated tissues was observed 20,21 . However, the development of CAII-positive type I/II oligodendrocytes in the spinal cord and molecules that regulate their development have not been elucidated yet.
Teneurin-4 (Ten-4), a type II transmembrane glycoprotein, has been identified as a regulator of myelination of small diameter axons 22 . Ten-4 is one of four teneurin family members in vertebrates and is expressed in glia cells including oligodendrocytes 22,23 . Our previous results with the electron microscopy (EM) analysis of 7-week-old mouse spinal cord revealed the defect of myelination in small diameter axons, while these axons were formed 22 . Further, the number of total oligodendrocytes, which were positive with the antibody CC1, was reduced in Ten-4 deficient (−/−) mice 22 . However, an investigation regarding types of oligodendrocytes, particularly type I/II oligodendrocytes, in Ten-4 −/− mice has not been carried out.
In this study, we examined the normal development of CAII-positive type I/II oligodendrocytes in the spinal cord and the association of Ten-4 with type I/II oligodendrocytes by immunohistochemistry and EM analysis. From our results using 7-week-old WT spinal cord, CAII-positive cells were found in the whole spinal cord, but were accumulated in the CST, FG, and the inside part of VF, in which small diameter axons existed. Besides, these cells emerged from postnatal day (P) 7 to 11. In Ten-4 −/− mice, CAII-positive type I/II oligodendrocytes were reduced in the WM. The reduction of these cells was already observed at P11. Our findings indicated that WT type I/II oligodendrocytes myelinate small caliber axons in the CST, FG, and the inside part of VF and their development is regulated by Ten-4. To our knowledge, this is the first report that identified a regulator of specific types (type I/II) of oligodendrocytes.

Results
Distribution and development of cAii-positive type i/ii oligodendrocytes around small caliber axons in the spinal cord. To examine the distribution of CAII-positive cells in the spinal cord, we first performed immunostaining in the cervical spinal cord tissues of 7-week-old WT mice using anti-CAII and anti-neurofilament (NF) antibodies to detect type I/II oligodendrocytes and axons, respectively ( Fig. 1). In the DC region of the WM, we could confirm that small diameter axons were located in the CST and FG, but larger axons were in the FC, by the immunostaining of NF (Fig. 1a,b,d). The intensity of the NF immunostaining was weak in both the CST and FG, compared with the other areas, because diameters of CST and FG axons were small. With higher exposure of NF immunofluorescence, NF-positive small diameter axons were observed ( Supplementary Fig. S1). Also, smaller axons were found in the inside region of the VF, more than in the outside VF region (Fig. 1b,c). Therefore, we subdivided the VF into 2 regions and hereafter designated the inside and outside regions VFi and VFo, respectively (Fig. 1a-c). As a result of CAII staining, CAII-positive type I/II oligodendrocytes were located both in the WM and GM (Fig. 1b). CAII is expressed not only in type I/II oligodendrocytes but also in satellite oligodendrocytes, which adjacently exist with neuronal soma in the GM and unmyelinate axons 20,24 . When we focused on the VF tissues of the WM, the higher number of CAII-positive cells was observed in the VFi than VFo (VFi: 500.8 ± 80.45 cells/mm 2 ; VFo: 151.5 ± 31.71 cells/mm 2 ) (Fig. 1b,c,e). In the DC, there were more CAIIpositive cells in the CST and FG, rather than FC (CST: 1,292 ± 191.5 cells/mm 2 ; FG: 495.1 ± 100.5 cells/mm 2 ; FC: 233.2 ± 97.87 cells/mm 2 ) (Fig. 1b,d,f), while a statistical difference was not obtained in the numbers between FG and FC (Fig. 1f). Also, many puncta of CAII staining, reminiscent of numerous well-branched thin processes that myelinate smaller axons, were observed in the CST and FG (Fig. 1d). Taken together, we found that there were many CAII-positive type I/II oligodendrocytes around the small caliber axons, especially in the VFi, CST, and FG of the WM of the spinal cord.
Reduction of type I/II oligodendrocytes in the spinal cord of Ten-4 −/− mice. In our previous EM analysis, small diameter axons were unmyelinated in the VF/LF of 7-week-old Ten-4 −/− mice 22 . Here, we tested immunostaining of myelin basic protein (MBP) in the cervical spinal cord of Ten-4 −/− mice, and found that the defects of myelin formation of small caliber axon were detected, especially in the CST and FG ( Supplementary  Fig. S2).
We then asked whether CAII-positive type I/II oligodendrocytes were normally developed or not in the Ten-4 −/− spinal cord. Therefore, we performed immunostaining of CAII in mouse tissues at 7 weeks. CAII was expressed in only type I/II oligodendrocytes in the WM and the antibody CC1 was used as a control marker for all types of oligodendrocytes. In the WT spinal cord tissue, about 55% of CC1-positive oligodendrocytes expressed CAII (CC1-single positive cells: 329.0 ± 74.22 cells/mm 2 ; CAII/CC1-double positive cells: 405.7 ± 80.95 cells/ mm 2 ) (Fig. 3a,c). There were no CAII-single positive cells (Fig. 3a,b). Most of CAII/CC1-double positive type I/ II oligodendrocytes were located particularly in the VFi, CST, and FG ( Fig. 3b: arrows). However, in the Ten-4 −/− spinal cord tissue, CAII/CC1-double positive type I/II oligodendrocytes were dramatically reduced, compared with WT, whereas CC1-single positive oligodendrocytes were also declined but a certain number of them was found in the Ten-4 −/− tissue (CC1-single positive cells: 141.5 ± 23.39 cells/mm 2 ; CAII/CC1-double positive cells: 4.208 ± 1.426 cells/mm 2 ) (Fig. 3a-c). Though CC1-single positive cells were reduced in a half in Ten-4  Fig. S3). The further observation with higher magnification revealed that the larger cell bodies of CC1-single positive cells in both WT and Ten-4 −/− mice were reminiscent to the characteristics of type III/IV oligodendrocytes ( Fig. 3b: arrowheads), but not type I/II oligodendrocytes (Fig. 3b: arrows). Hence, Ten-4 is required for either production or maintenance of CAII-positive type I/II oligodendrocytes in the spinal cord.
Onset of the myelination defect of small diameter axons. Though the hypomyelination of small caliber axons in Ten-4 −/− mice was reported in our previous study 22 , it has not been investigated during the postnatal stage. Therefore, we finally examined whether the myelination of small diameter axons in Ten-4 −/− mice was already interrupted during the postnatal stage or not, due to the reduction of type I/II oligodendrocytes. We assessed the relation of myelination and axonal caliber sizes by immunostaining of MBP and NF at P3, P7, and P11, when myelination began in the WT spinal cord. MBP positive myelinated axons were counted with their diameters. As a result, no differences between WT and Ten-4 −/− tissues were observed in myelination of both small and large diameter axons at P3 (Fig. 5a,b). Larger diameter axons (0.8 μm < ) were preferentially myelinated at this stage. However, at P7, the percentages of myelinated axons were decreased only in smaller diameter axons (<1.2 μm) but not in large diameter axons (1.2 μm < ) in the Ten-4 −/− tissue, compared to WT (Fig. 5a,b). This defect became more obvious at P11 (Fig. 5a). Furthermore, we performed an electron microscopy analysis of the spinal cord tissues at P7. In WT mice, the myelination of both small and large diameter axons was observed (Fig. 5c). By contrast, in Ten-4 −/− mice, myelination of small diameter axons was rarely found at this stage, although large axons were myelinated (Fig. 5c). These results suggest that Ten-4 positively regulates the formation of myelin around the small diameter axons at the beginning of myelination during the early postnatal stage. Together, we conclude that Ten-4 is responsible for the generation of type I/II oligodendrocytes, which myelinate small diameter axons during the initial stage of myelination in the spinal cord.

Discussion
Our results in this report showed that first, CAII-positive type I/II oligodendrocytes were preferentially located in the small caliber axon areas and initiated to appear from P7 to P11 in the WT spinal cord. Second, we found that type I/II oligodendrocytes were diminished in the Ten-4 −/− spinal cord. Furthermore, the reduction was already started at P11. These results suggest that small caliber axons are myelinated by CAII-positive type I/II oligodendrocytes in the spinal cord during the oligodendrocyte differentiation stage and Ten-4 is required for the generation of type I/II oligodendrocytes for the myelination of small diameter axons.
In our previous study, we found that the differentiation of oligodendrocyte precursor cells (OPCs) into oligodendrocytes and their cell survival were inhibited in Ten-4 −/− mice at P6, although the type of oligodendrocytes was unknown 22 . At the stage, the NF expression and staining as a marker for axon formation was normal in the Ten-4 −/− spinal cord 22 . In the present study, the myelination of the small diameter axons was already inhibited at P7, before high expression of CAII in oligodendrocytes at P11. Around these stages, OPCs interact with axons and receive axonal signals, which trigger their differentiation into oligodendrocytes 25 . In addition, OPCs/ oligodendrocytes that fail to adhere and receive the axonal survival factors die through apoptosis [26][27][28] . Indeed, we recently demonstrated that the extracellular domain of Ten-4 promoted OPCs/oligodendrocytes adhesion and differentiation 29 . From these observations, we speculate that OPCs are unable to interact with smaller axons properly and fail to differentiate into type I/II oligodendrocytes with the expression of CAII in the Ten-4 −/− spinal cord tissue. Additionally, OPCs whose fate is determined into type I/II oligodendrocytes are possibly eliminated through apoptosis before the expression of CAII.
Recently, a couple of groups identified that the lack of expression or function of cell adhesion molecules, such as focal adhesion kinase, β1 integrin, and its ligand laminin α2 chain, caused the defect of myelination in small caliber axons 30,31 . Moreover, cell adhesion is also important for the expression of CAII. In the report by Kida and her colleagues, they demonstrated that CAII was localized in oligodendrocyte processes as well as cell bodies, and CAII-positive varicosities, which were accumulated alongside oligodendrocyte processes, were substantially were observed in the CST than in the FC. Though the number in the FG was also larger than in the FC, there was not the quantitative difference. Triplicate experiments were independently performed (n = 3). Error bars represent mean ± s.e.m. One-way ANOVA followed by Tukey's post hoc test for multiple comparisons was used for the statistical analysis. *p < 0.05, **p < 0.01.

Scientific RepoRtS |
(2020) 10:8611 | https://doi.org/10.1038/s41598-020-65485-0 www.nature.com/scientificreports www.nature.com/scientificreports/ reduced without the contact with axons 24 . In our study, the reduction of CAII expression including punctuated signals and hypomyelination in small diameter axons were observed in the spinal cord tissue of 7-week-old mice. In addition, our recent report proved that Ten-4 on oligodendrocytes promoted cell-cell adhesion via bindings with teneurins (Ten-1 to Ten-4) on axons, which positively regulated myelin formation 29 . Ten-4 may regulate the cell adhesion to small caliber axons for the differentiation/maturation of CAII-positive type I/II oligodendrocytes.
Hypomyelination and axon degeneration in small caliber axons of the spinal cord are observed in some myelin-related disorders. In Pelizaeus-Merzbacher disease (PMD) patients with the null mutation in the proteolipid protein 1 gene (PLP1), which is a major myelin protein as well as MBP, axon degeneration was observed in the CST and FG 32 . Furthermore, in MS patients, small diameter axons in the CST and FG were degenerated 4,5 . Pathological character of MS in acute phase is that the cycle from demyelination to remyelination is repeated in the various CNS tissues 8 . Interestingly, according to the autopsy samples from acute MS patients, CAII-positive cell number and the expression level of CAII were transiently increased, compared with those from the healthy control samples, though oligodendrocytes positive for the other marker CNPase were not changed. The increased CAII-positive cells may be associated with the activated remyelination of small diameter axons 20 . The other report showed that central pontine myelinolysis (CPM) patients displayed the remarkable upregulation of CAII expression in oligodendrocytes 21 . In this study, we found that the signal of CAII expression in Ten-4 −/− mice at P11 was temporally intensified, in spite of little number of CAII-positive cells. From these evidences, it is possible that Ten-4 may be involved in the pathological conditions of these de-or dys-myelinating diseases through the regulation of CAII-positive type I/II oligodendrocytes. Ten-4 could be a marker in the pathogenesis of these diseases.
Myelin structure of small caliber axons plays important roles for not only body movements but also cognitive functions. Indeed, a MRI analysis revealed that the defect of small caliber axons was observed in the brain of MS patients, whose symptom was cognitive impairments 7 . From the recent interesting studies, chronic oral administration of D -aspartate (D-Asp) improved the function of memory, nevertheless the mechanism has not been www.nature.com/scientificreports www.nature.com/scientificreports/ clarified yet 33 . Also, AMPA receptor agonists promoted the myelination and oligodendrocyte differentiation 34 . D-Asp treatment increased the small caliber axonal activity, so that the activity dependent remyelination selectively occurred 35 . In conclusion of these reports, the promoted activity in the small caliber axons by the AMPA www.nature.com/scientificreports www.nature.com/scientificreports/ receptor agonists or D-Asp may be useful for the therapy for these myelin-related disorders including cognitive deficiency [35][36][37] . Though the association of type I/II oligodendrocyte with these mental disorders has not been demonstrated, our present report may shed light on the relation.
In summary, we could identify Ten-4 as a regulator of type I/II oligodendrocytes. To our knowledge, Ten-4 is the first responsible regulator of specific types (type I/II) of oligodendrocytes. Because the neurological and mental functions in the CNS are regulated by various sizes of myelinated axons, the identification of regulating molecules of myelination depending on axonal sizes is necessary. Importantly, Ten-4 has been already identified as the associated gene with neurological and mental diseases, such as essential tremor, bipolar disorder, and schizophrenia [38][39][40] . Our findings will contribute to the elucidation of the molecular mechanisms for understanding oligodendrocyte biology and may be useful for the development of diagnostic or therapeutic reagents in the related diseases.

Methods
Mice. The Ten-4 −/− mouse line (furue) that we characterized previously 22 was kindly provided by Dr.
Yoshihiko Yamada from NIDCR, NIH. Littermates at P3, P7, P11, and 7 weeks were used for experiments. All procedures for experimental animals were approved by the Institutional Animal Care and Use Committees of Tokyo Medical and Dental University (Protocol No: 017028C). All methods were conducted in strict accordance with the approved guidelines of the institutional animal care committees.
Immunohistochemistry. For the preparation of spinal cord tissue sections, P3, P7, and P11 mice were perfused with 5 ml of phosphate buffered saline (PBS) and following fixed with 5 ml of 4% paraformaldehyde (PFA, Wako) in PBS. Fifteen ml of PBS and 15 ml of 4% PFA were used for perfusion of mice at 7 weeks. The spinal cords were dissected out and post-fixed with 4% PFA in PBS. After the post-fixation overnight at 4 °C, the vertebra surrounding the spinal cord tissue at P11 and 7 weeks was removed, spinal cord tissues were soaked into 15% sucrose in PBS, and placed overnight at 4 °C. Then, the tissues were put into 30% sucrose in PBS for 2 days www.nature.com/scientificreports www.nature.com/scientificreports/ at 4 °C, and were frozenly embedded with O.C.T compound (Sakura Finetek) on dry ice. After that, the spinal cord tissues were sliced into 8 μm sections with cryostat (Leica LCM). The tissue sections were dried for 1 to 2 h, and the sections were fixed with 4% PFA in PBS for 10 min at room temperature and were washed with PBS 3 times (excessive fixation caused a reduction in the reactivity of anti-CAII antibody). To retrieve the antigen, the sections were heated with Target Retrieval Solution, Citrate pH 6.1 (Dako) by microwave. After blocking with Power Block Universal Blocking Reagent (BioGenex Laboratories) for 1 h at room temperature, primary antibodies, rabbit anti-CAII (1:250; Sigma-Aldrich), mouse anti-APC (clone CC1) (1:500; Millipore), rabbit anti-MBP (1:100; Millipore), and rabbit anti-neurofilament (1:500; Sigma-Aldrich), in 1% bovine serum albumin (BSA) in