YAP and TAZ regulate remyelination in the central nervous system

Myelinating cells are sensitive to mechanical stimuli from their extracellular matrix. Ablation of YAP and TAZ mechanotransducers in Schwann cells abolishes the axon–Schwann cell recognition, myelination, and remyelination in the peripheral nervous system. It was unknown if YAP and TAZ are also required for myelination and remyelination in the central nervous system. Here we define the importance of oligodendrocyte (OL) YAP and TAZ in vivo, by specific deletion in oligodendroglial cells in adult OLs during myelin repair. Blocking YAP and TAZ expression in OL lineage cells did not affect animal viability or any major defects on OL maturation and myelination. However, using a mouse model of demyelination/remyelination, we demonstrate that YAP and TAZ modulate the capacity of OLs to remyelinate axons, particularly during the early stage of the repair process, when OL proliferation is most important. These results indicate that YAP and TAZ signaling is necessary for effective remyelination of the mouse brain.

modulators, as well as extrinsic signals such as growth factors, hormones, and mechanical cues (reviewed in Tsai & Casaccia, 2019).
Mechanical cues can activate YAP and TAZ, transcription coactivators of the Hippo pathway.When YAP and TAZ are phosphorylated, they are inactive.Upon activation, YAP and TAZ are dephosphorylated and shuttle from the cytosol to the nuclei and interact with transcription factors of the TEAD family (reviewed in Feltri et al., 2021).In the PNS, YAP and TAZ are both essential for axon-Schwann cell recognition, proliferation, and myelination (Deng et al., 2017;Fernando et al., 2016;Grove et al., 2017;Poitelon et al., 2016).More specifically, YAP and TAZ promote myelination by regulating transcription of myelin proteins and lipid synthesis regulators, synergistically with SOX10 and EGR2 (Deng et al., 2017;Fernando et al., 2016;Grove et al., 2017;Lopez-Anido et al., 2016;Poitelon et al., 2016;Wu et al., 2018).In addition, YAP and TAZ also play a role in peripheral nerve regeneration and remyelination following nerve crush injury (Grove et al., 2020;Jeanette et al., 2021;Mindos et al., 2017).
In the CNS, many studies have shown that YAP and TAZ regulate the self-renewal and maintenance of neural stem cells (Bao et al., 2017;Cao et al., 2008;Ding et al., 2016;Gil-Ranedo et al., 2019;Han et al., 2015;Lavado et al., 2013;Lavado et al., 2018;Van Hateren et al., 2011;Yao et al., 2014).In addition, YAP was shown to be expressed by OLs in vitro (Jagielska et al., 2017;Ong et al., 2020;Shimizu et al., 2016;Urbanski et al., 2016).Shimizu et al. showed that overexpression of YAP in OLs decreased the number of mature OLs in vivo.The authors also demonstrated that knock-down of YAP in OLs limits the extension of primary processes in vitro, suggesting that it impairs their capacity to myelinate (Shimizu et al., 2016).Ong et al. showed in vitro that YAP is a mediator of mechanical cues from the environment in myelinating OLs, but not in differentiating OL precursor cells (OPCs) (Ong et al., 2020).Finally, animals ablated for both Yap and Taz in adult myelinating Schwann cells and OLs (Yap f/f ; Taz f/f ; Plp1-Cre ER ) exhibit severe weight loss, tremors, ataxia, and mortality within 2 weeks (Deng et al., 2017;Grove et al., 2017;Grove et al., 2020;Jeanette et al., 2021).However, preliminary exploration by Deng et al. did not detect overt myelin abnormalities in the adult corpus callosum or spinal cord of these animals.Thus, while YAP and TAZ respond to mechanical stress and impact OLs differentiation in vitro, there is a need to clarify if YAP and TAZ modulate in vivo OL lineage cell proliferation, maturation, myelination, and remyelination.
In this study, we used two different genetic mouse models to selectively inactivate YAP and TAZ (Yap f/f ; Taz f/f ; Olig2-Cre and Yap f/f ; Taz f/f ; Plp1-Cre ER ) to clarify the function of YAP and TAZ in developing (Olig2-Cre) and myelinating OLs (Plp1-Cre ER ).We also studied the role of YAP and TAZ in myelination and remyelination following cuprizone treatment.Supported by morphological, cellular, and molecular data, we show here that ablation of YAP and TAZ has no major effect on myelination.However, we found that ablation of YAP and TAZ in OL lineage cells significantly affects OPC proliferation and delays the remyelination process, suggesting that YAP and TAZ are required in the OPCs of the adult brain to properly remyelinate the CNS.

| Animal models
This study was carried out in accordance with the principles of the Basel Declaration and recommendations of ARRIVE guidelines issued by the NC3Rs and approved by the Albany Medical College Institutional Animal Care and Use Committee (no.20-08001).Yap f/f and Taz f/f mice (Reginensi et al., 2013) were derived to a congenic C57BL/6J background (as described in Poitelon et al., 2016), bred with Plp1-Cre ER (Jackson Laboratory, #005975) (Doerflinger et al., 2003) or Olig2-Cre mice (Jackson Laboratory, #025567) (Zawadzka et al., 2010) to generate Yap f/f ; Taz f/f ; Plp1-Cre ER (Jeanette et al., 2021) and Yap f/f ; Taz f/f ; Olig2-Cre.Genotyping of mutant mice was performed by PCR on tail genomic DNA (Poitelon et al., 2016).
Tamoxifen (Sigma-Aldrich, T5648) was injected into Yap f/f ; Taz f/f ; Plp1-Cre ER mice.A 20 mg/mL solution of tamoxifen was prepared in autoclaved corn oil.This solution was injected intraperitoneally at a concentration of 100 mg/kg body weight starting on post-natal day 40 (P40).A total of five injections were performed, spaced apart every 12 h.The resulting mutant mice were compared with their littermates injected with corn oil only, and to Yap f/f ; Taz f/f animals injected with tamoxifen.Animals were housed in cages of five with 12/12 h light/ dark cycles.No animals were excluded from the study.Equal numbers of males and females were included in the study.Mutant and control littermates from both sexes were culled at the indicated ages, and optic nerves and brains were collected.

| Cuprizone treatment in mice
Postnatal day 40 (P40) mice were fed with powder chow, freshly prepared with 0.2% copper chelator agent Cuprizone (Sigma-Aldrich, C9012) for 6 weeks.Half of each experimental group was randomly selected and culled after 6 weeks of the cuprizone demyelination period to analyze YAP and TAZ function during demyelination.The other half were back to normal diet and allowed to recover for up to 4 weeks with normal pellet chow to analyze YAP and TAZ function during remyelination.Body weight of mice was closely monitored prior and during the 6 weeks of cuprizone treatment.

| Electron microscopy
Mutant and control littermates were euthanized at the indicated ages, and optic nerves were dissected.Optic nerves were fixed in 2% buffered glutaraldehyde and post fixed in 1% osmium tetroxide.
After alcohol dehydration, the samples were embedded in EPON resin.Ultrathin sections (80-85 nm thick) were stained with saturated uranyl acetate and lead citrate and examined by electron microscopy for morphometry analysis.For all morphological assessments, at least three animals per genotype were analyzed.For g-ratio analysis of optic nerves (axon diameter/fiber diameter), ultrathin section images were acquired with an FEI Tecnai F20 transmission electron microscope (TEM).Axon and fiber diameters were quantified from TEM images using the ImageJ software (imagej.nih.gov/ij).G-ratios were determined for at least 100 fibers chosen randomly per animal.
Images from cultures OLs were acquired using a Leica confocal microscope, model SP5II.Images for quantifications from brains, spinal cords, and optic nerves were acquired using the Axio observer fluorescent microscope (Carl Zeiss Microscopy, Jena Germany) The staining intensity for myelin proteins and the number of positive cells were determined at a depth of 1.1 mm to bregma into the central area of the corpus callosum (0.6 mm 2 ), in the motor cortex area (0.6 mm 2 ) and in the striatum (0.6 mm 2 ).The integrated fluorescence intensity was calculated as the product of the area and mean pixel intensity using ImageJ (http://imagej.nih.gov/ij).All quantification of positive cells and fluorescent intensity results were determined from at least four brains per experimental group.

| Statistical analysis
Experiments were not randomized, but data collection and analysis were performed blind to the conditions of the experiments.Data were analyzed using GraphPad Prism 6.01.Data are presented as mean ± standard error of the mean (S.E.M.).No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field.Two-tailed Student's t-test was used for statistical analysis of the differences between two groups.Values of pvalue ≤ .05were considered to represent a significant difference.

| YAP and TAZ are expressed in the CNS and OLs
To clarify the role of YAP and TAZ in OLs, we first looked at YAP and TAZ expression level in the CNS.We investigated both the corpus callosum and optic nerve, two regions of the CNS rich in OLs and myelin.In developing optic nerve, we found that YAP and TAZ were expressed at low level between postnatal day 6 (P6) and P10, when OLs start myelinating; then YAP and TAZ expression increased from P15 up to P90.In comparison, in developing corpus callosum, YAP and TAZ are stably expressed from P6 to P90.These data indicate that YAP and TAZ are expressed in CNS regions rich in OLs (Figures 1a and S1).In agreement with those observations, we detected YAP and TAZ expression in vitro in both OPCs and mature OLs, with YAP and TAZ localization being nuclear as well as cytoplasmic (Figures 1b and S2).
To further investigate the role of YAP and TAZ in OLs in the adult CNS, we used the double knockout mouse model Yap f/f ; Taz f/f ; Plp1-Cre ER .Plp1-Cre ER expression is conditional following intraperitoneal injections of tamoxifen and allows for temporally controlled ablation of YAP and TAZ in adult myelinated glial cells (both OL lineage and Schwann cells).This mouse model is only viable for about 14 days following tamoxifen injection, which limits the window of analysis for the study of YAP and TAZ function in the CNS (Deng et al., 2017;Grove et al., 2017;Grove et al., 2020;Jeanette et al., 2021).Yap f/f ; Taz f/f ; Plp1-Cre ER animals received 5 injections of tamoxifen, one every 12 h at postnatal day 40.We analyzed YAP and TAZ protein levels in optic nerve 13 days after tamoxifen injection.Only YAP protein levels were visibly reduced in Yap f/f ; Taz f/f ; Plp1-Cre ER (Figures 1c and S1), suggesting that TAZ is also expressed by other CNS cells, such as astrocytes and endothelial cells (Zhang et al., 2014).Collectively these data support that both YAP and TAZ are expressed in OLs in the adult CNS.However, as only YAP protein levels are significantly affected in the Yap f/f ; Taz f/f ; Plp1-Cre ER mouse model, the data suggest a high proportion of non-OL cells express TAZ protein in the brain.

| YAP and TAZ are not essential for OL maturation and myelination in the CNS
Because the Yap f/f ; Taz f/f ; Plp1-Cre ER mouse model is only viable for about 14 days following tamoxifen injection (Deng et al., 2017;Grove et al., 2017;Grove et al., 2020;Jeanette et al., 2021), the window of analysis for the study of YAP and TAZ function in the CNS was limited to 13 days post recombination.Thus, the role of YAP and TAZ in OLs was evaluated by immunohistochemistry, in animals at 53 days of age (P40 plus 13 days after tamoxifen injection).Compared with control littermates, the fluorescent signal for MBP was not reduced in the corpus callosum, cortex, striatum, optic nerves, and spinal cord of Yap f/f ; Taz f/f ; Plp1-Cre ER animals (Figures 2 and S3).Also, the length and thickness of the corpus callosum were not significantly different in Yap f/f ; Taz f/f ; Plp1-Cre ER animals, and their controls (Figure 2c).The myelination of the Yap f/f ; Taz f/f ; Plp1-Cre ER CNS was also evaluated by immunohistochemistry for myelin OL glycoprotein (MOG) and proteolipid protein1 (PLP1).No significant differences were observed for these proteins in the brain (Figure S4).To analyze the effect of YAP and TAZ deletion on OLs, the total number of OL lineage cells (OLIG2-positive cells) was analyzed in the brain, spinal cord, and optic nerve.The total number of OLIG2-positive cells was unaffected by the absence of YAP and TAZ in OLs in all CNS areas (Figure 2a and S3).
The myelination of the Yap f/f ; Taz f/f ; Plp1-Cre ER animals was also assessed by electron microscopy.The degree of myelination was analyzed by calculating the g-ratio of myelinated axons in optic nerve (Figure 3).The Yap f/f ; Taz f/f ; Plp1-Cre ER animals did not show any changes in the mean g-ratio of myelinated axons (Figure 3b) or in the average diameter of myelinated axons (Figure 3c).
To further investigate the role of YAP and TAZ at an earlier time point in brain development, we used the double knockout mouse model Yap f/f ; Taz f/f ; Olig2-Cre, which allows site-specific recombination in all OL lineage cells and motor neurons, as early as embryonic day 10.5 (Dessaud et al., 2007;Zawadzka et al., 2010).Importantly, compared to Yap f/f ; Taz f/f ; Plp1-Cre ER animals, the Yap f/f ; Taz f/f ; Olig2-Cre animals did not display any weight loss, tremors, ataxia, and mortality.Myelination was evaluated by immunohistochemistry at 7, 20, 40, and 60 days of age.Compared with control littermates, no significant differences were observed in the brain of Yap f/f ; Taz f/f ; Olig2-Cre for the fluorescent signal for MBP, and in the number of OLIG2-positive cells or the dimensions of the corpus callosum at 20 days of age (Figure 4) as well as at 7, 40, and 60 days of age (Figure S5).In addition, no differences were observed for the fluorescent signal for MBP and the number of OLIG2-positive cells in optic nerve (Figure S6).Overall, the results obtained with Yap f/f ; Taz f/f ; Plp1-Cre ER and Yap f/f ; Taz f/f ; Olig2-Cre animals suggest that YAP and TAZ are not essential for OL maturation and myelin maintenance in the CNS.

| YAP and TAZ are required for the remyelination of the adult mouse brain
To determine how the loss of YAP and TAZ affects the remyelination potential of OLs in the adult mouse brain, we used the cuprizone model of demyelination and remyelination.We used P60 control (Yap f/f ; Taz f/f ) and Yap f/f ; Taz f/f ; Olig2-Cre mice, which did not present obvious myelination defects (Figure S5).Mice were treated with cuprizone for 6 weeks to induce demyelination.
Brains were then collected for analysis after 6 weeks of cuprizone treatment and at 4 weeks of recovery in normal diet.Six weeks of cuprizone treatment induced a severe demyelination in the brain of both control and Yap f/f ; Taz f/f ; Olig2-Cre mice, as well as weight loss (Figures 5 and S7).A significant recovery in MBP, PLP, and MOG levels was observed in the corpus callosum and cortex of both control and Yap f/f ; Taz f/f ; Olig2-Cre after the 4 weeks of remyelination following withdrawal of cuprizone treatment (Figure 5).Yet Yap f/f ; Taz f/f ; Olig2-Cre mice showed significantly less MBP, PLP, and MOG production than control mice in the cortex and in the lateral corpus callosum suggesting a delay in remyelination (Figure 5).These reductions were also observed by Western blot of myelin proteins (Figure 5).We detected a decrease in the number of OLIG2-positive cells and OLIG2-positive cell proliferation during recovery in the corpus callosum and cortex of Yap f/f ; Taz f/f ; Olig2-Cre mice (Figure 6), but not in the number of mature OLs (CC1-and OLIG2-positive cells) (Figure 6).Interestingly, absence of YAP and TAZ in OLs appears to have a more severe effect in the corpus callosum than in the cortex (Figure 6).We did not observe any increase in apoptosis in OL lineage in Yap f/f ; Taz f/f ; Olig2-Cre after the 2 weeks of remyelination following withdrawal of cuprizone treatment (data not shown).Collectively these data support that expression of both YAP and TAZ are required for the remyelination

| DISCUSSION
Although remyelination occurs, this process cannot be sustained and eventually fails in CNS myelin disorders such as progressive multiple sclerosis.Thus, understanding how the OLs integrate cues necessary for proper remyelination is critical to develop new means to treat patients with myelin disorders.Our knowledge regarding the integration of mechanical stimuli during remyelination has primarily come from in vitro models.In contrast, the conditional knockout mice described in this work allowed us to specifically study the in vivo consequences of YAP and TAZ deficit in the OL lineage during myelination and remyelination.

| YAP and TAZ in myelinating glial cells
The biological role of YAP and TAZ is cell, tissue, and biological context specific.They can have broad effects, as seen for Schwann cell proliferation, differentiation, myelination, and remyelination (Deng et al., 2017;Fernando et al., 2016;Grove et al., 2017;Grove et al., 2020;Jeanette et al., 2021;Poitelon et al., 2016).Schwann cells share numerous similarities with OLs.However, there are important differences between OLs and Schwann cells in their embryological origin (i.e., neural stem cells for OLs vs. neural crest cells for Schwann cells), development, mechanisms of wrapping, molecular composition, response to injury, and the distinct signaling pathways and transcription factors that regulate their differentiation and myelin formation (e.g., OLIG1/2 and MYRF in OLs vs. POU3F1 and EGR2 in Schwann cells) (reviewed in Herbert & Monk, 2017).In accordance with these differences, diseases of myelin are in general restricted to myelinated fibers in the CNS or in the PNS (e.g., Multiple Sclerosis, leukodystrophies in the CNS vs. Charcot-Marie-Tooth disease, Guillain-Barré Syndrome in the PNS).Most importantly some promyelinating pathways in the PNS have an opposite role on myelin formation in the CNS (e.g., MLC2, KIF13B, DNM2) (Gerber et al., 2019;Noseda et al., 2013;Wang et al., 2008).Therefore, despite YAP and TAZ being critically needed for Schwann cell biology, there was an outstanding gap to assess the function of YAP and TAZ in OLs and to determine if, like in the PNS, YAP and TAZ are also required for CNS myelination and remyelination.Our data show that, despite being expressed in the OL lineage, YAP and TAZ seem to be dispensable for myelin maintenance in the CNS.While we cannot exclude that ablation of YAP and TAZ in the developing brain of young animals could have an effect of OL proliferation, survival, and differentiation, their ablation had no major effect of OL maturation and myelination.Yet, distinctively during repair, our data also show that ablation of YAP and TAZ significantly delays the remyelination process, suggesting that YAP and TAZ are required in the OL lineage of the adult brain to properly proliferate and myelinate the CNS after demyelination.It will be important for future studies to investigate these mechanisms in depth.First, the defect in proliferation seen in OL lacking YAP and TAZ could come from a defect in the proliferation of OL lineage, or from a defect in the proliferation or migration of neural stem cells (Heng et al., 2020).Second, we observed the absence of YAP and TAZ in OLs seems to affect more severely the OL proliferation in the corpus callosum compared to the cortex, thus suggesting a region-specific function for YAP and TAZ, with a more important role in controlling OL proliferation in whiter matter than in gray matter.Finally, while YAP and TAZ regulate myelin formation in the PNS through TEAD1 and EGR2 (Lopez-Anido et al., 2016), it is still unknown which transcriptional factors are partnering with YAP and TAZ to regulate remyelination.

| YAP and TAZ regulation in OLs
OLs like other cells in the CNS, are subjected to both tension forces (mediated by the cytoskeleton) and compression forces (mediated by the extracellular matrix and neighboring cells) during development and aging, and in pathological conditions (reviewed in Makhija et al., 2020).Numerous works have demonstrated that mechanical cues regulate myelinating glial cell migration, differentiation, proliferation, and myelination (Acheta, Bhatia, et al., 2022;Fernando et al., 2016;Hernandez et al., 2016;Jagielska et al., 2012;Jagielska et al., 2017;Kim et al., 2020;Lourenco et al., 2016;Ong et al., 2020;Poitelon et al., 2016;Poitelon et al., 2017;Rosenberg et al., 2008;Urbanski et al., 2016;Urbanski et al., 2019).In addition, changes in extracellular matrix deposition and composition may highly contribute to changes in mechanical properties and brain remyelination capacity, as demonstrated by prior work showing that acute demyelinated lesions following a 6-week cuprizone treatment are softer than healthy brain tissue (Urbanski et al., 2019).
Several studies have shown the involvement of constitutive signaling pathways such as YAP and TAZ to transduce mechanical signals into transcriptional regulation in OLs (Jagielska et al., 2017;Ong et al., 2020;Shimizu et al., 2016;Urbanski et al., 2016).In addition, proper amount of YAP and TAZ activation is paramount to regulate OL biology during development as both in vitro YAP knockdown and in vivo YAP overexpression can decrease the formation of OL processes (Shimizu et al., 2016).
Here, our work indicates that two mechanotransducers YAP and TAZ are also required for OL lineage proliferation and the remyelination of the CNS following cuprizone-induced demyelination, but not for OL maturation or differentiation.These results are somewhat surprising given that (i) a stiffer environment promotes the translocation of YAP to the nucleus in response to both tensile strain (Shimizu et al., 2016) and rigid substrates (Ong et al., 2020;Urbanski et al., 2016); (ii) acutely demyelinated brain is softer than healthy brain tissue (Urbanski et al., 2019).Yet, while mechanical cues may contribute to the regulation of YAP and TAZ, YAP, and TAZ are regulated by non-mechanical signals from the HIPPO signaling pathways (Dobrokhotov et al., 2018;Feltri et al., 2021).In addition, it is possible that the functions of these other mechanosensitive pathways in OLs could predominate over YAP and TAZ in regulating the OL lineage (Hernandez et al., 2016;Segel et al., 2019;Velasco-Estevez et al., 2020).
In sum, there are more efforts needed to understand the mechanobiology of OLs.First, it is essential to better characterize the contributors to the changes in mechanical environment, to OL mechanosensation, and mechanotransduction in diseases.Second, further studies should focus on characterizing these contributors in vivo or if done in vitro using materials at a stiffness within the pathophysiological range.Finally, it will be important to understand how OLs integrate mechanical cues together with biochemical signals.
are expressed by mouse oligodendrocytes (OLs).(a) (top) Western blot analysis of YAP and TAZ in wildtype optic nerve (left) and corpus callosum (right) at 6, 10, 15, 21 and 30 days of age in mouse.Calnexin (CNX) was used as loading control.(bottom) Relative quantification of YAP and TAZ protein level from densitometries.(b) Immunolocalization of YAP and TAZ in OPC (SOX10 + ) (kept in proliferation conditions) and OL (PLP + ) mouse cultures show localization of YAP and TAZ in OL precursor cells (OPC) and OL nuclei and cytoplasm.Scale bar, 10 μm.(c) Western blot analysis of YAP and TAZ on optic nerve from Yap f/f ; Taz f/f ; Plp1-Cre ER animals, 13 days after treatment with tamoxifen, at 53 days of age.YAP protein levels are lower in Yap f/f ; Taz f/f ; Plp1-Cre ER injected with tamoxifen compared to control Yap f/f ; Taz f/f ; Plp1-Cre ER .CNX was used as loading control.n ≥ 3 mice for each condition.Protein expression data were normalized to the average of the control.Data are presented as mean ± S.E.M. Two-tailed Student's t test: **, p-value ≤ .01.
protein level and OLIG2-positive cell number are not affected in Yap f/f ; Taz f/f ; Plp1-Cre ER brain.(a) (left) Representative coronal sections of brain tissue immunostained for MBP and OLIG2 collected from adult control and Yap f/f ; Taz f/f ; Plp1-Cre ER animals, 13 days after treatment with tamoxifen, at 53 days of age.Scale bar, 200 μm.(right) Integrated fluorescence intensity for MBP and the number of OLIG2-positive cells were quantified in the lateral corpus callosum (CC), the cingulate cortex (CX) and striatum (ST); n ≥ 6 mice for each condition.(b) CC length and midline thickness from adult control and Yap f/f ; Taz f/f ; Plp1-Cre ER animals, 13 days after treatment with tamoxifen, at 53 days of age; n ≥ 8 mice for each condition.Intensity data were normalized to the average of the control for each brain region.Data are presented as mean ± S.E.M.
ER optic nerve.(a) Electron micrographs of axons in the optic nerve of control and Yap f/f ; Taz f/f ; Plp1-Cre ER mice, 13 days after treatment with tamoxifen, at 93 days of age.Scale bars, 2 μm.(b) Scatter plot of g-ratio values of myelinated axons.(c) Mean axonal diameter of myelinated axons.n > 100 fibers per animal were analyzed.Data are presented as mean ± SD.F I G U R E 4 MBP protein level and OLIG2-positive cell number are not affected in Yap f/f ; Taz f/f ; Olig2-Cre brain.(a) (left) Representative coronal sections of brain tissue immunostained for MBP and OLIG2 collected from adult control and Yap f/f ; Taz f/f ; Olig2-Cre animals, at 20 days of age.Scale bar, 200 μm.(right) Integrated fluorescence intensity for MBP and the number of OLIG2-positive cells were quantified in the lateral corpus callosum (CC), the cingulate cortex (CX) and striatum (ST); n ≥ 4 mice for each condition.(b) CC length and midline thickness from adult control and Yap f/f ; Taz f/f ; Olig2-Cre animals, at 20 days of age.n ≥ 4 mice for each condition.Intensity data were normalized to the average of the control for each brain region.Data are presented as mean ± S.E.M.

F
I G U R E 5 Remyelination is delayed in the adult Yap f/f ; Taz f/f ; Olig2-Cre brain.(a) Schematic representative of the cuprizone treatment as well sampling timepoints.(b) Representative coronal sections from control and Yap f/f ; Taz f/f ; Olig2-Cre brains immunostained for MBP.Tissues were collected from untreated, control, and Yap f/f ; Taz f/f ; Olig2-Cre mice at the end of the cuprizone (CPZ) treatment and after 1 week (1 W Rec), 2 weeks (2 W Rec), or 4 weeks of recovery (4 W Rec). Scale bar, 200 μm.(c) Integrated fluorescence intensity for MBP, MOG, and PLP were quantified in the cortex (cx) of untreated, control, and Yap f/f ; Taz f/f ; Olig2-Cre mice at the end of the CPZ treatment and after 1, 2, and 4 weeks of recovery.Fluorescent intensity data are presented as percent of untreated control mice.(d) Western blot analysis of MBP and MOG on cortex from control and Yap f/f ; Taz f/f ; Olig2-Cre animals, 1 or 2 weeks after CPZ withdrawal.MBP and MOG protein levels are lower in Yap f/f ; Taz f/f ; Olig2-Cre, after 1 week of recovery.GAPDH was used as loading control; n ≥ 3 mice for each condition.IHC intensity data were normalized to the average of the untreated control.WB intensity data were normalized to the average of the control for each brain region.Data are presented as mean ± S.E.M. Two-tailed Student's t test: *, p-value ≤ .05;**, p-value ≤ .01;****, p-value ≤ .0001. in the CNS, and for proliferation of OL precursors during remyelination.

F
I G U R E 6 Oligodendrocyte proliferation is reduced in the corpus callosum of Yap f/f ; Taz f/f ; Olig2-Cre animal during remyelination.(a) The number of OLIG2-positive, and CC1-positive cells were quantified in the corpus callosum and cortex of control, and Yap f/f ; Taz f/f ; Olig2-Cre mice at the end of the CPZ treatment and after 1, 2, and 4 weeks of recovery.(b) The number of KI67-positive cells were quantified in the corpus callosum of control, and Yap f/f ; Taz f/f ; Olig2-Cre mice after 2 weeks of recovery; n ≥ 3 mice for each condition.CC1+/Olig2+ and KI67+/Olig2+ data are represented as percentage of Olig2+ cells.Data are presented as mean ± S.E.M. Two-tailed Student's t test: *, p-value ≤ .05;****, p-value ≤ .0001.