Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

Introduction

Thyroid hormones (THs) are essential regulators of growth and development in various tissues, including the brain [1,2]. Most of these processes occur through binding to specific nuclear receptors known as TH receptors or TRs. They function as hormone-activated transcription factors that control the expression of a large number of target genes. For these genomic actions, thyroxine or T4 acts primarily as a prohormone while T3 is the major ligand that binds to different TR isoforms [2,3]. During neurodevelopment, THs participate in neuronal proliferation, migration, differentiation and signaling, as well as in brain myelination [4]. Indeed, TH deficiency during the mammalian fetal or early postnatal periods may cause irreversible mental retardation and neurological deficits [5,6]. Furthermore, the hypothyroid brain presents delayed myelination and poor deposition of myelin, whereas hyperthyroidism during the early postnatal period results in accelerated myelination [7-10].

Myelin is an axonal insulator that facilitates the propagation of action potentials and myelination is the last major event to occur during central and peripheral nervous system development of all vertebrates except agnathans [11-13]. In mammals, myelination begins in late stages of embryonic development and ends in the postnatal stages [4] while in the zebrafish (Danio rerio) it starts around 2.5 days post fertilization (dpf) [14-16]. This is why the zebrafish has become a popular model organism to uncover novel mechanisms controlling vertebrate myelination [17,18] and therefore also its TH dependency.

The fundamental structure of myelin is conserved in vertebrates. Homologs for myelin proteins such as myelin basic protein (MBP), myelin protein zero (MPZ), and proteolipid protein (PLP1) are present in zebrafish. It is currently accepted that these three structural proteins are distributed in both the central- (CNS) and the peripheral nervous systems [17,19]. In the CNS, neural precursor cells give rise to oligodendrocyte precursor cells (OPCs), which proliferate as they migrate through the developing CNS. Following migration, OPCs transition into pre-myelinating oligodendrocytes (OLs) which upon terminal differentiation become myelinating OLs and iteratively wrap their plasma membranes around multiple axon segments to form myelin sheaths [16]. Nascent myelin sheaths are evident around 2.5 dpf in the zebrafish CNS [18,20]. By 3 dpf, myelin is detected in the ventral hindbrain and robustly myelinated bundles of axons are observed by 7 dpf [17,21], extending rostrally over the following days to the cerebellar plate, midbrain, and optic nerve [22]. The transcription factors olig1/2 control OL linage progression in early stages and subsequently, OL differentiation [14]; olig2 transcripts are present in the embryo [23] and protein expression starts around 9 hours post-fertilization (hpf) in the ventral region of the neural plate [12]. The transcription factor sox10 is specifically required for survival of cells committed to myelination and its expression starts around 10.5 hpf in neural precursors [16]. Among zebrafish myelin-related genes, at least olig2, mbp and mpz have been shown to be direct T3-responsive genes [24], like their mammalian counterparts [25-27]. Thus, it is possible that the stimulatory effects of THs on myelination first lead to OL maturation and then to the synthesis of myelin structural proteins by myelinating OLs; both events would involve genomic paths mediated by TRs.

During the first days post-fertilization in teleost embryos, including zebrafish do not synthetize TH but the fertilized eggs contain a load of T4, which is gradually converted to T3 by the activity of deiodinases [28]. Here, we blocked conversion of maternal and later of larval T4 to T3 through treatment with IOP, a drug proven to inhibit deiodinase activity in mammals as well as in non-mammalian vertebrates, including teleosts [29-31] to analyze the effects of the induced T3 deficiency on myelination. Using myelin- and OPC staining techniques as well as transgenic mbp:egfp zebrafish larvae, we here show that T3 deficiency reduced the amount of myelin in the analyzed brain areas, possibly by reducing the amount of OPCs, as suggested by a lower NG2 immunoreactivity.

Materials and methods

Zebrafish

Wild-type zebrafish embryos obtained from crosses of adult male and female zebrafish were used, collected at the one cell stage and kept under standard conditions at 28°C in E3 embryonic medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4 and methylene blue), with 14:10 h light:dark cycles. After 4 dpf, larvae were fed twice a day with an interval of at least 6 hours between meals. During each feeding, the larvae were in contact with the food flake for 1 hour, and then the E3 medium was replaced. This study was carried out in strict accordance with the national and international guidelines for the care and use of laboratory animals. The protocol was approved by the Ethics for Research Committee of the Instituto de Neurobiología of the Universidad Nacional Autónoma de México (Protocol Number: 095A). Euthanasia was performed through hypothermal shock by immersion in ice water (2–4°C), and all efforts were made to minimize suffering.

Quantitative PCR

RNA was extracted in control and treated groups with Trizol Reagent (Life technologies). Retrotranscription was performed with M-MLV Reverse Transcriptase (Promega) from 500 ng of total RNA and 0.5 μg oligo (dT). The used oligonucleotides () were as previously published [23] and designed with real time PCR tool from IDT (Integrated DNA Technologies). Primer specificity was confirmed by agarose gel electrophoresis and melting curve analysis. In all cases, reactions contained 1 μL of reverse transcribed product, Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), and oligonucleotides at a final concentration of 0.5 μM. A Step One instrument was used for detection and data analysis according to the manufacturer’s instructions (Applied Biosystems). For all genes, amplicons obtained by RT-PCR were cloned (pJET1.2/blunt Cloning Vector; Thermo Scientific) and sequenced. These constructs were used to prepare standard curves ranging from 101 to 109 molecules/μl. Absolute quantifications were obtained interpolating the Ct value of each experimental sample in the standard curve of the corresponding gene and normalizing with reference genes β-actin and LSM. The absolute mRNA concentration was expressed as molecules per microgram of total mRNA used in the RT reaction. Identical PCRs from the RNA samples before the reverse transcription reaction yielded no detectable products.

Alcian blue staining

Alcian blue staining was performed in zebrafish larvae as previously reported [32]. Briefly, 7 dpf larvae were anesthetized, immersed overnight in PFA 4% and washed in PBS 0.1% Tween-20. Animals were bleached in 30% hydrogen peroxide until eye depigmentation was evident. Again, embryos were washed in PBS-Tween and transferred into 1% of hydrochloric acid, 70% ethanol, and 0.1% Alcian blue overnight. Finally, samples were cleared in acidic ethanol for 4 hours, dehydrated in ethanol series and stored in glycerol. Pictures were captured with a Leica DM500 microscope at 10X and LAS EZ 3.0 software.

Myelin staining with Black-Gold II

Fixation of zebrafish larvae was performed immediately after euthanasia by immersion in freshly prepared 4% paraformaldehyde (PFA) and then incubated overnight at 4°C. The next day the larvae were washed 5 times with 1x PBS for 5 min at room temperature (RT). Subsequently, they were cryoprotected with 10, 20 and 30% sucrose solutions in water and incubated overnight again. Whole-mount Black-Gold II staining was performed using a modified protocol [33], in which the larvae were briefly rehydrated with 1x PBS for 2 min at RT; then incubated in 0.3% Black-Gold II solution at 60°C until staining was complete and afterwards were washed 5 times with 1x PBS for 2 min at 60°C. Finally, larvae were dehydrated with a graduated series of alcohol solution, rinsed with xylene, and stored at -20°C in Eppendorf tubes.

For image analysis, each zebrafish larva was embedded in 1.5% low melting point agarose, examined in sagittal position and Rostral-Caudal (R-C) orientation with the LEICA microscope objectives of 4x and 40x and with the integrated ICC50 HD color camera. Zebrafish brain 40x images were analyzed with Image-Pro Plus 6 (IPP6) as follows: myelin cords stained in violet color with Black-Gold II [34] were selected and the density of myelin fibers was quantified based on the number of positive pixels in the selected area. IPP6 allows to select a specific color (violet) in the tinted image, but without considering their intensity. All regions were selected manually.

OPC staining

Immunostaining was performed according to [35] using primary antibodies shown to recognize fish NG2 [36]. Briefly, zebrafish larvae were fixed overnight with PFA 4%, dehydrated in methanol series and stored at -20°C. For immunostaining, larvae were rehydrated, washed with PBS and incubated in a 3% H2O2/0.5% KOH medium for 40 min to decrease pigmentation. To unmask antigens, larvae were incubated in sodium-citrate (pH 6.0) for 30 min. Additionally, larvae were submerged in acetone 100% for 30 min, washed with sterile water for 5 min and incubated with a blocking solution (PBS triton 1% plus BSA 0.1%) for 30 min. The primary antibody anti-NG2 (1:100, Millipore, USA, Cat. no. AB5320) was incubated for two days at 4°C. The secondary antibody (goat anti-rabbit IgG, 1:250, Invitrogen, USA, Cat. No. A11008) was incubated overnight at 4°C.

For image capture, zebrafish larvae were oriented dorsally onto 1.5% low melting point agarose in concave slides and the top of the head was microphotographed. Fluorescence was detected with a Carl Zeiss-LSM 700 confocal microscope and ZEN software. The images were obtained at 10x and were scanned three-dimensionally at a 114 μm depth in set Z-scan (0.66 pixels) at a resolution of 512 × 512 pixels. The Image J program from the Fiji package was used for data processing.

mbp:egfp transgenic fish

To generate embryos that express egfp under the mbp promoter, we used the Tol2 transposon system [37]. To this end, transposase mRNA was synthetized in vitro using a Not1-linearized pCS-zT2TP plasmid (gift from Dr. K. Kawakami) as template in an mMESSAGE mMACHINE SP6 kit (Life Technologies). Transposase mRNA was purified by ethanol precipitation and resuspended in RNase-free water. One cell stage F0 zebrafish embryos were injected [38] no later than 20 minutes after fertilization, with a volume of 1 nL of working solution containing 25 pg of transposase mRNA and 25 pg of plasmid Tg (mbp:egfp) [39]. These embryos were collected and submitted to IOP treatment (0.5, 1, 1.5 and 2 μM) for 3 dpf. The same approach has been used previously to visualize GFP expression in F0 zebrafish (e.g. [40,41]). To take into account the variability due to the inherent mosaicism in F0 transgenic fish, we analyzed at least 15 animals per group allowing to show clear effects of the treatments.

After fixation (see above), fluorescence was detected as described for OPC staining with the following changes: The images were obtained at 20x and zebrafish heads were scanned three-dimensionally at a 260 to 300 μm depth in set Z-scan (0.66 pixels) at a resolution of 512 × 512 pixels. The area of interest was manually segmented, and the intensity threshold was matched for all the scanned images. The heads of the larvae were reconstructed using the Amira-Avizo software for 3D visualization and the values were reported in voxels.

IOP treatment and T3 rescue

We first tested different doses of IOP to determine the lowest effective concentration (). For further experiments, wild-type larvae were subjected to one of the following 4 treatments: solvent control, IOP 0.5 μM, T3 10 nM and IOP 0.5 μM + T3 10 nM. These concentrations were added to the E3 medium and were replaced daily for 3 or 7 days. Since long-term (7 dpf) treatment with T3 resulted in 100% mortality of the larvae, only Ctrl and IOP conditions were analyzed at this stage. Myelin density was quantified following Black-Gold II staining and the mRNA expression of the genes involved in the myelination process was measured. As an additional way to visualize the changes in myelin in response to T3 deficiency and restoration, we also subjected mbp:egfp F0 zebrafish embryos to the above mentioned treatments and analyzed the fluorescent signal. Image analysis and data processing were performed as described above.

Statistical analysis

Statistical tests were carried out using GraphPad Prism 7 using appropriate tests as specified in each figure legend. The difference was considered statistically significant at p <0.05.

Results

Optimization of IOP treatment duration and concentration

We first quantified expression of myelin-related genes (olig2, sox10, mbpa, mpz) at various stages of development of the complete zebrafish embryo (0 hpf) and 3, 4, 5 and 7 dpf larvae (). We found expression of all but mpz at 0 hpf, suggesting that these early transcripts are of maternal origin [23]. For all analyzed genes, larval expression was stable after 3 dpf. Given that myelination processes have commenced by 3 dpf and continue during larval development, we chose 3 and 7 dpf as stages for further detailed analysis.

We next treated zebrafish larvae up to 7 dpf with 0.5, 1, 1.5 and 2 μM IOP to determine the minimum concentration of IOP that would change the expression of these genes. Since all doses clearly had an impact on gene expression () we opted for the lowest dose of 0.5 μM IOP for all further experiments to minimize possible toxic effects.

Effects of IOP treatment on thyroid status

To corroborate that the IOP treatment was inducing T3 deficiency, we first used Alcian staining, confirming a malformation in Meckel´s cartilage, a characteristic of a hypothyroid status in mammals and fish [42,43], in 7 dpf IOP-treated animals (). We then analyzed the expression of genes that are involved in maintaining the thyroidal status [deiodinase type 2 (dio2) and deiodinase type 3 (dio3)] as well as genes that are known to be TH-dependent [growth hormone (gh), thyrotropin (tshb) and transthyretin (ttr)]. As expected, IOP treatment induced an up- (dio2) and down- (dio3) regulation of the T3-activating and inactivating pathways, respectively (). gh and ttr expression did not respond to the IOP treatment while that of tshb was significantly increased. To corroborate that the selected genes were indeed regulated by THs, we treated 3 and 7 dpf larvae with T3 (10 nM) and analyzed their expression levels. As previously mentioned, the long-term (7 dpf) treatment with T3 resulted in 100% mortality of the larvae. However, treatment in 3 dpf larvae induced dio3 and tshb gene expression changes in the opposite way as compared with IOP-treated larvae. Moreover, gh and ttr expression increased when exposed to a surplus of T3, demonstrating that TH availability does influence the regulation of these genes (). Given the cartilage malformation and discrete responses observed following treatment with 0.5 μM IOP, our results point to a mild hypothyroidism/T3 deficiency.

Effects of IOP treatment on thyroid status.

(A) Effect of IOP on Meckel´s cartilage morphology. Left: Representative image of Alcian blue staining of 7 dpf control zebrafish (left) and IOP-treated (right) larvae. Scale bar 100 μm. Right: Graph depicting the quantification of Meckel’s cartilage (n = 3 larvae per group). The difference is best visible in the top middle where the stained area is thinner and more faint in the IOP animal. Scale bar 100 μm. Statistical analysis was performed with a Student’s t test, *p<0.01 (B) Changes in expression of TH-regulated genes in zebrafish larvae exposed to IOP. mRNA quantification of dio2, dio3, tshb, ttr and gh in controls and larvae exposed to IOP 0.5 μM or T3 10 nM for 3 dpf and to IOP 0.5 μM for 7 dpf. Data are represented as individual values in vertical graphs (n = 5 pools from independent experiments of 50–60 larvae per pool and condition). Graphs are showing individual values and mean ± SEM. Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the corresponding control groups. Significant differences are indicated as *p <0.05, **p <0.01, and ***p <0.001.

Quantification of myelin with Black-Gold II staining

By analyzing specific regions in the forebrain [dorsal telencephalon (D), dorsal thalamus (DT)], midbrain [tectum opticum (TO)], hindbrain [cerebellar plate (CeP) and spinal cord tract (SCT)] in 3 dpf (Ctrl, IOP, T3 and IOP + T3) ( and 7 dpf (Ctrl vs IOP) zebrafish larvae ( with whole-mount Black-Gold II staining we identified myelinated axons in the form of longitudinal filaments in violet color. We measured the density of the myelin sheaths and detected a strong reduction in the larvae treated with IOP at 3 and 7 dpf (Figs and ). The myelinated area (MA) quantified in pixels in [D + DT + TeO], CeP and SCT (Figs and ) was significantly reduced in IOP-treated larvae, both at 3dpf and 7dpf. Treatment with T3 alone did not change myelin density compared to control, but addition of T3 to the IOP treatment partly rescued the decrease observed with IOP alone.

Whole mount Black-Gold II staining of zebrafish larvae at 3 dpf.

(A) Side view painting of 3 dpf zebrafish larva showing D + DT + TeO; CeP and SCT location; (B) Schematic drawing of the brain region (forebrain, midbrain and hindbrain) with identification of myelinated longitudinal axons in a violet color. (C) Representative photomicrograph of zebrafish larvae with lateral view and R-C orientation, whole-mount Black-Gold II staining in D + DT + TeO; CeP and SCT at 3 dpf (Unstained CTRL, Solvent Ctrl/BG-II, IOP/BG-II, T3/BG-II and IOP + T3/BGII). In each corner, the amplification of sagittal sections identifying the selected area for analysis and the positive myelin pixels, recognized by the IPP6 program; scale bar: 500 μm and 50 μm. (D) Quantification of myelin density expressed as myelinated area (MA) in pixels2 in D + DT + TeO; CeP and SCT in CTRL, IOP, T3 and IOP + T3 larvae (mean ± SEM with n = 5/condition). Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the control group. *p <0.05, **p <0.01, ***p <0.001.

Whole-mount Black-Gold II staining of zebrafish larvae at 7 dpf.

(A) Photomicrograph with lateral view and R-C orientation, whole-mount Black-Gold II staining in D + DT + TeO; CeP and SCT at 7 dpf (Unstained CTRL, Solvent CTRL/BG-II and IOP/BG-II), scale bar: 500 μm and 50 μm. In each corner, the amplification of sagittal sections identifying the selected area for analysis and the positive myelin pixels, recognized by the IPP6 program; (B) Graph showing the quantification of myelin density expressed as myelinated area (MA) in pixels2 (individual values and mean ± SEM with n = 5/condition). Statistical analysis was performed with Student’s t test. ***p<0.001, ****p <0.0001.

Expression of myelin-related genes

As a mean to verify that the observed effects upon myelination were indeed due to the lack of T3, we performed a rescue experiment including the following experimental groups: Ctrl, IOP (0.5 μM), T3 (10 nM) and IOP (0.5 μM) + T3 (10 nM) and analyzed the expression of genes involved in myelination in 3dpf larvae. As mentioned earlier, 7 dpf T3-treated larvae did not survive. IOP treatment resulted in a decreased expression of olig2, mpz and plp1b and T3 treatment up regulated their expression at 3 dpf (). sox10 expression increased at 3 dpf in IOP-treated larvae and decreased with T3, while expression of mbpa only decreased after 7 days of IOP treatment but was enhanced by T3 in 3 dpf larvae ().

Changes in expression of myelin-related genes in zebrafish larvae exposed to IOP.

mRNA quantification of olig2, sox10, mbpa, mpz and plp1b in controls and larvae exposed to IOP 0.5 μM, T3 10 nM or IOP 0.5 μM + T3 10 nM for 3 dpf and to IOP 0.5 μM for 7 dpf. Data are represented as individual values in vertical graphs (n = 5 pools from independent experiments of 50–60 larvae per pool and condition). Graphs are showing individual values and mean ± SEM. Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the corresponding control groups. Significant differences are indicated as *p <0.05, **p <0.01, and ***p <0.001.

Visualization of myelin in mbp:egfp zebrafish larvae

As an additional way to visualize the changes in myelin in response to T3 deficiency and restoration we also performed experiments on mbp:egfp transgenic animals. We first analyzed mbp:egfp signal (voxels) in D + DT + TeO and CeP of 3 dpf zebrafish larvae exposed to increasing concentrations of IOP (0.5, 2 and 5 μM). As the effect was already clear at IOP 0.5 μM (), we continued with this concentration for a rescue experiment and analyzed the mbp:egfp signal in 3 dpf zebrafish larvae exposed to T3 (10 nM) without or with IOP (0.5 μM). As shown in and 5E, the fluorescence emitted by the myelinated regions again decreased when the larvae were treated with IOP. However, T3 supplementation to IOP larvae increased the fluorescent signal which was restored to levels similar to those of the control group. exposes the fluorescence inside the brain of zebrafish larvae with a 360° rotation.

Fig 5

IOP and T3 rescue treatment in mbp:egfp transgenic zebrafish larvae at 3 dpf.

(A) Lateral scan of the head using confocal microscopy and generating multiple 3D optical sections; scale bar: 100 μm. (B) Representative photomicrograph of zebrafish larvae with dorsal view and R-C orientation, whole-mount Black-Gold II staining in D, DT and at 3 dpf (wild-type, CTRL, IOP, T3 and IOP + T3). scale bar: 50 μm. (C) Schematic drawing with dorsal view of the head; the asterisks represent signal in the habenula and between the telencephalon (Tel) and tectum opticum (TeO) (red); signal within the TeO (white) and signal in the dorsal Tectum and between TeO and cerebellar plate (CeP) (yellow). The black arrow represents the orientation of the larvae, rostral-caudal (R-C). (D) The images are the representation of the maximum intensity projection of the entire set of Z-stacks: uninjected CTRL (WT), CTRL, larvae treated with IOP 0.5 μM, T3 10 nM or IOP 0.5 μM + T3 10 nM; scale bar: 50 μm. (E) Graphs depicting the volume of the myelinated area in voxels, Data are shown as individual values and mean ± SEM (approximately n = 15 larvae/group). Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the control group. **p< 0.01 and ***p< 0.001.

OPC development

In order to analyze if T3 deficiency was delaying OPC maturation, we immunostained 3 and 7 dpf control and IOP-treated larvae with the OPC marker NG2. As shown in , NG2 label is stronger in 3 dpf control larvae, as compared to 7 dpf controls, suggesting a gradual OPC to OL differentiation. IOP treatment reduced the amount of NG2 stain in 3 dpf larvae, as compared to controls, while 7 dpf controls and IOP-treated larvae were no longer significantly different. To further analyze a possible myelination delay in vivo, myelin fluorescent signal was followed in living larvae at the onset of myelination (2.5 dpf) and once myelin formation is ongoing (3 dpf) using mbp:egfp transgenic larvae with or without IOP treatment. As shown in , no myelin was detected in 2.5 dpf controls, while it was clearly visible at 3 dpf. In contrast, 2.5 and 3 dpf IOP-treated larvae did not show a myelin signal.

OPC immunostaining.

Dorsal view of the head of zebrafish larva at 3 and 7 dpf. (A) Pictures show whole mount NG2 immunoreactivity in control (upper panel) and IOP-treated larvae (lower panel). The latter show significantly decreased immunoreactivity (B) Negative control (without primary antibody) (C) Quantification of total fluorescence intensity of NG2 immunoreactivity. Individual values and the mean ± SEM are shown (n = 5 larvae/group). Statistical analysis was performed using Student’s t test. Significant differences are indicated as **p< 0.01. Scale bar 100 μm.

Discussion

Thyroid hormones are key modulators of myelination in vertebrates. Hyperthyroidism during the postnatal period results in accelerated myelination, while delayed myelination and poor deposition of myelin is observed in individuals with congenital hypothyroidism [4,10]. In the present study we implemented a protocol that mimics this condition by reducing the conversion of T4, initially of maternal origin, to bioactive T3 and thus limiting its availability during the early stages of neurodevelopment. In the present study we induced a moderate T3 deficiency which allowed to investigate the impact of mildly reduced T3 signaling without too strongly disturbing overall development of the larvae.

When analyzing myelin content in the CNS using Black-Gold II staining, we observed a clear myelin reduction as early as 3 dpf, which became more evident after 7 dpf. A delay in development secondary to IOP treatment could explain myelin reduction; however, we did not observe differences in the length of 3 dpf control and IOP-treated larvae (3.206 ± 0.017 mm2), suggesting that at that stage there was no overall developmental delay and hypomyelination was a specific response to lower T3 bioavailability. Of note is the fact that a decrease in total length was observed in 7 dpf IOP-treated larvae (3.933 ± 0.015 mm2) as compared to controls (3.813 ± 0.027 mm2). Thus, as expected, a longer exposure to reduced T3 impairs both, growth, and myelination. The reduction in the amount of myelin in IOP-treated zebrafish larvae is in line with observations in developing hypothyroid rats, where a clearly lower number of myelinated axons in the anterior commissure and the corpus callosum was described [44]. Another mammalian study reported that hypothyroid neonatal rats had the same number of myelinated axons as control animals but revealed a decrease in the area of these axons [45]. Although more detailed analysis, for instance by electron microscopy, would be needed to draw firm conclusions, data from the present study coincide with studies in which myelin deposition is affected during prenatal and neonatal T3 deficiency [4]. The observed reduction in CNS myelin content did not seem to greatly affect movement, since no differences between control and IOP-treated 7dpf larvae zebrafish were observed, at least not in natural swimming behavior (data not shown).

In the present study the hypomyelination shown by Black Gold II staining was additionally confirmed using mbp:egfp transgenic animals. We observed a clear reduction in fluorescence in the brain of 3 dpf larvae when T3 availability was low. In concert with the reduction in fluorescence, changes in myelin-related gene expression were also observed. Keeping in mind that analyses were performed on whole larvae RNA and therefore only global net effects could be detected, the results point to a clear effect of T3 deficiency on myelin-related gene expression.

It is known that the onset of myelination starts with OPC formation, which depends at least in part on the expression of transcription factors olig1/2 and sox10, and is followed by mature myelin sheath formation, for which genes involved in myelin composition are expressed (mbp, mpz and plp1) [18]. Additionally, sox10 is required for the survival of myelinating OLs [16,20]. THs promote OPC differentiation, migration and maturation by increasing expression of Olig2 and Sox10 genes in mammals [46]. In the zebrafish, THs up-regulate the expression of genes encoding structural myelin proteins (mbp and mpz) and some of the myelination-related genes (olig2, mbp and mpz) were recently shown to contain TH-responsive elements in their promoter regions, as previously shown in mammals [24]. In the present study, 3 dpf IOP-treated larvae showed decreased expression of olig2, mpz and plp1b, genes that are constitutively expressed in CNS mature OL, pointing to a delay in myelination possibly due to a reduced number of mature OL, secondary to TH deficiency, as previously reported in both zebrafish [24,47] and mammals [48,49]. Furthermore, we also observed a peak of expression of sox10 at 3 dpf in IOP-treated larvae, suggesting a possible compensatory mechanism to maintain the reduced number of committed myelinating OLs following T3 deficiency.

The hypomyelination observed in IOP-treated mbp:egfp transgenic animals was restored after T3 supplementation. These results agree with our previous observations in methimazole-treated juvenile tilapia where the thickness of myelinated axons in the cerebellar granular layer was reduced and could be restored by T3 treatment [50]. T3 rescue also reversed the changes in myelin-related gene expression induced by IOP exposure, evidencing that THs are involved in the molecular pathways regulating myelin formation. The significant increase in olig2, mbpa and mpz and the reduction in sox10 expression might indicate that the rescue T3 concentration we used was rather high. Given that myelination is established by 3 dpf, an excess of T3 could have a deleterious effect upon myelin-related gene expression. These observations provide further evidence of the importance of TH action during specific time windows in development, and prompt for additional studies to find out whether early exposure to excess T3 only accelerates or possibly disarrays myelin formation.

So far, the specific molecular mechanisms involved in TH-mediated myelination are far from clear. However, and even when THs regulate myelin structural proteins, their effects seem to initially target the differentiation and fate of neural precursor cells destined for myelin formation. Our observations in live mbp:egfp larvae showing that T3 deficiency delayed the onset of myelination suggest that OPC number was reduced and/or that myelin synthesis was altered in OLs. The fact that the immunoreactivity for the OPC marker NG2 was also reduced in 3 dpf IOP-treated larvae suggests T3 deficiency could arrest or delay neural progenitor to OPC transition and/or inhibited OPC radial proliferation and migration.

In conclusion, TH deficiency decreased the amount of myelin in different areas of the CNS and affected the expression of myelin-related genes. Since myelination/demyelination/remyelination are dynamic TH-dependent processes, more specific studies are required to better understand the factors involved in OL differentiation and maturation. Established developmental models such as zebrafish are crucial for future avenues of research to address these questions and related demyelination pathologies.

Expression of olig2, sox10, mbpa and mpz at the onset and establishment of myelin formation during zebrafish development.

A. mRNA quantification at 0 hpf, 3dpf, 4dpf, 5dpf and 7dpf. Results are represented on a logarithmic scale (mean ± SEM; n = 3 pools of 50–60 larvae per stage). B. 7 dpf zebrafish larvae were exposed to 0.5 μM, 1 μM, 1.5 μM and 2 μM IOP and mRNA expression of olig2, sox10, mbpa and mpz genes was quantified. Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the control groups. Significant differences are indicated as *p <0.05, **p <0.01, ***p <0.001 and ****p<0.0001.

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mbp:egfp expression in transgenic zebrafish larvae exposed to IOP.

Schematic drawing with dorsal view of the head. The dashed lines mark the brain of the larva (green) and the eyes (white). Red and yellow asterisks refer to the regions that make the division between Tel: Telencephalon, TeO: Tectum opticum, CeP: Cerebellar plate and MO: Medulla oblongata. Confocal images of the larval brain without treatment and with IOP 0.5, 2 and 5 μM treatment are shown. The images are the representation of the maximum intensity projection of the entire set of Z-stacks. (B) The graphs show the volume of the myelinated area in voxels. Data are shown as individual values and mean ± SEM (approximately n = 13 larvae/group). Statistical analysis between medians was performed using one-way ANOVA coupled with Tukey’s multiple comparison test. Significant differences (CTRL vs Treatment) are indicated as *p< 0.05, ***p< 0.001 and ****p< 0.0001.

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In vivo confocal imaging.

Control and IOP 0.5 μM-treated mbp:egfp transgenic zebrafish were photographed at 2.5 dpf and 3 dpf. At each time point, larvae were anesthetized with Tricaine mesylate (MS-222) 0.2 mg/ml for 45 seconds and fixed and orientated in agarose 1.5%. Confocal images were captured as previously indicated. After caption, animals were returned in a new chamber to their respective treatment. Given that capture was performed in live larvae, the orientation of the head was not always optimal.

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Real time PCR oligonucleotide sequences used to amplify the different genes.

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3D reconstruction with Amira software of mbp:egfp signal in 3dpf zebrafish larval brain.

Dorsal view of the head and brain of zebrafish larvae during the development of brain myelination. The mbp:egfp signal (represented in yellow) is located between the telencephalon and the TeO as well as between the TeO and the CeP. Upper images: Compilation of multiple data from 3D optical sections with signal mbp:egfp of control and IOP 0.5 μM treated zebrafish larvae; scale bar: 50 μm. Lower images: Zebrafish larvae with mbp:egfp signal, treated with T3 10 nM, and IOP 0.5 μM + T3 10 nM.

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3 Feb 2021

PONE-D-20-33696

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination

PLOS ONE

Dear Dr. Orozco,

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1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In “Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination,” Farías-Serratos et al. test the role of thyroid signaling in oligodendrocyte myelination. This question is of clinical impact, as human hypo/hyperthyroidism is linked to changes in myelination state. The authors use pharmacology to manipulate thyroid signaling and use a combination of qPCR, transgenic, and histological analyses of myelinating glial cell development. While the implications of this study are interesting and the manuscript is well written, I have several major concerns about the interpretation of the results that would necessitate further investigation before publication. Overall, I am concerned with the Black-Gold stains for assessing myelin in the larvae, as they have so much background that it is difficult to interpret what is the true signal. Below are suggestions for alternatives to improve my confidence in the results:

MAJOR:

1. Assessing oligodendrocyte development by qPCR is difficult to interpret because a number of the markers assessed are expressed in large populations of other cell types: olig2 in motor neurons, sox10 in all neural crest, mbpa in Schwann cells. The authors should take advantage of preexisting transgenic lines or published in situ constructs for these markers, which would be more suitable to address a developmental delay in oligodendrocytes specifically.

2. While the difference in Black-Gold II stain in Figure 2 is obvious, it is impossible to see the difference in the spinal cord (Figure 3) due to the extensive background staining. The authors should perform cross sections of the stained tissue and quantify the signal specifically within the spinal cord.

3. The Black-Gold stain in Figure 4 has so much background that I do not think it actually reflects myelination. At 3 dpf, there is very little myelin present in the brain, so I do not feel confident that the authors are actually quantifying myelin. The authors should take advantage of published in situ probes for mbpa, or rely solely on the mbp:egfp transgene.

4. Assessing oligodendrocyte myelination in single oligodendrocytes through injection of mbp:egfp for mosaic labeling would be useful to determine whether there is a developmental delay (delayed transition to making myelin sheaths), or if fewer oligodendrocytes are being made in general.

5. Given the morphological defects in the IOP-treated animals, the authors should use acetylated tubulin stain to assess whether axon number and/or organization is impaired in these animals.

MINOR:

Line 67: Myelin is not present in all vertebrates. The sentence should be clarified to state that myelination is the last major event to occur in all vertebrates for which axons are myelinated.

Lines 77-79: Mbp is also robustly expressed in Schwann cells. The major difference between central and peripheral myelin is the presence of Plp in oligodendrocytes and Mpz in Schwann cells.

Lines 338-339: Olig2 is not specific to oligodendrocyte precursors. It is also a primary marker of motor neurons (PMID: 12167410), which complicates the interpretation of the qPCR results as noted above.

Statistics: Why were medians compared rather than means?

Reviewer #2: The article by Farias-Serratos et al describes the consequences of pharmacological treatment with iopanoic acid (IOP) on the myelination of zebrafish larvae. The authors treated zebrafish larvae with different concentrations of IOP (and chose the smallest one 0.5 µM) and show deregulation of key genes for thyroid hormone signalling (dio2, dio3, thra, thrb) and oligidendrocyte maturation (olig2, mbp, mpz) in whole embryos. The authors show differences in myelin density with Black Gold II labelling in areas of the central nervous system such as the flat cerebellar or spinal cord tract (SCT) at 3dpf or 7 dpf. However, no significant changes are shown in the diameter or perimeter of the myelinated axons in the regions of the dorsal thalmamus or the ceP.

Rescue experiments are being carried out with T3 on previously deregulated genes ( but not on sections) and mbp :eGFP transgenic animals have been generated to make fluorescence quantification in the presence of IOP and with T3 rescue.

The article is clear and well written. Different parts are well balanced. Discussion is mainly based on a comparison of the data provided in the present article with the data published by Zada on mct8 -/- and could be improved.

I think the article needs minor corrections before publications in PLOS ONE.

1) rationale for the parts of the nervous system is not clear.

2) Even if IOP is known to affect deiodinase activity and thyroid hormoen synthesis , an embryonic treatment with IOP in zebrafish is not well linked with hypothyroidism. As hypothyroidism is not fully demonstrated, some conclusions are not supported by the data presented. Would it be possible to better show the hypothyroidism ?. Can T3 and T4 measurements be performed on embryos (whole or tissue) before and after IOP treatment to support the fundamental hypothesis of hypothyroidism? If not, can a T4 antibody be used? Or study of the thyroid gland? pituitary (TSH) or thyroid growth related genes?

3) The article is mostly descriptive and lacks a functional part to make the data fully usable and understandable.

4) Statistics: with n=3 for the gene exppressions for exemple. A comparison of more than two groups by mann whitney is not recommanded (ANOVA should be used but I don’t know if n=3 is enough for statistical power).

Following are some suggestions that can be taken into account to improve the MS according with editor’s recommandation.

Genes are quantified and related to a total amount of RNA. However, the actin B1 gene is in the list of primers used. It is not clear whether this housekeeping gene has been used for relative quantification or not.

The authors assume that the decrease in dio3 is a sign of hypothyroidism but the increased expression at 3dpf of thrb, mpa at 7 days and the decrease in thraa and thrab do not support this hypothesis. It is also debatable whether these measurements are made on whole organisms. Only an overall net effect is therefore observed and correlations with tissue or even cellular events in the nervous system are hazardous, given that opposite cellular regulations can be observed, particularly during development. A discussion on tissue and cellular effects of thyroid hormone would be therefore interesting

it is quite surprising to see that the T3 treatment alone has not been carried out on gene quantification and it is not described whether the 50/60 embryos x3 come from 3 independent experiments or if they are from the same experiment.

There is no functional experience on the consequences of a delay in myelination (movement for example) and rescue that the T3 treatment could bring. Furthermore, cross sections show a lack of effect of IOP treatment on myelinated axons. How then can this apparent contradiction be explained with the drop in myelin density observed with Black gold II marking? What are the effects of T3 and IOP+T3 treatment on these parameters?

Fig 6/ Video Suppl 4 : the term positive control is not appropriate (solvant control). In the video the size is not the same than the others 3D reconstructions.

A scale bar should be present in all sections/pictures/video

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Reviewer #2: No

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11 May 2021

Answers to Review

We thank the reviewers for their valuable comments and suggestions. In the new version of the manuscript, we have included additional experiments to address some of the concerns raised, as well as rewritten sections of the discussion. The changes are shown in red throughout the revised manuscript.

Reviewer #1:

1. Assessing oligodendrocyte development by qPCR is difficult to interpret because a number of the markers assessed are expressed in large populations of other cell types: olig2 in motor neurons, sox10 in all neural crest, mbpa in Schwann cells. The authors should take advantage of preexisting transgenic lines or published in situ constructs for these markers, which would be more suitable to address a developmental delay in oligodendrocytes specifically.

and

4. Assessing oligodendrocyte myelination in single oligodendrocytes through injection of mbp:egfp for mosaic labeling would be useful to determine whether there is a developmental delay (delayed transition to making myelin sheaths), or if fewer oligodendrocytes are being made in general.

We agree with the reviewer that qPCR is not the optimal way to follow up on oligodendrocyte development, considering also that the concentration of the target gene is diluted when mRNA is extracted from the whole embryo. In order to gain some insights regarding a possible delay in oligodendrocyte development due to T3 deficiency, we have included two additional experiments: 1) a follow-up on myelin formation using live mbp:egfp larvae at the onset of myelination and once myelin formation is ongoing; 2) immunostaining using an OPC marker to analyze if OPCs showed a developmental delay with T3 deficiency or if fewer OPCs transitioned to myelinating OLs. (lines 318-330 and 398-406).

2. While the difference in Black-Gold II stain in Figure 2 is obvious, it is impossible to see the difference in the spinal cord (Figure 3) due to the extensive background staining. The authors should perform cross sections of the stained tissue and quantify the signal specifically within the spinal cord.

We thank the reviewer for the comment. We have repeated the experiment in both, whole-mount preparations and cross-sections. The Black-Gold II staining was improved, resulting in images where myelin is more clearly observed; however, when we performed the Black-Gold II staining in cross-section, images were not optimal to accurately measure myelin content because sectioning harms the integrity of the tissue. Since the new whole-mount images depict myelin content quite unequivocally, we decided to just include these in the new version of the manuscript. Also, we have eliminated the Figure 3 from the original version of the manuscript and have included spinal cord images in the new Figures 2 and 3.

3. The Black-Gold stain in Figure 4 has so much background that I do not think it actually reflects myelination. At 3 dpf, there is very little myelin present in the brain, so I do not feel confident that the authors are actually quantifying myelin. The authors should take advantage of published in situ probes for mbpa, or rely solely on the mbp:egfp transgene.

The reviewer is right in that it is difficult to observe myelin in the cross-sections included in Figure 3 of the original version of the manuscript although the magnitude of magnification used while performing myelin determinations allowed the measurements reported in the Figure. However, given the comment and considering that no changes were observed in the analyzed parameters (perimeter and diameter), we decided to eliminate these data from the new version of the manuscript.

5. Given the morphological defects in the IOP-treated animals, the authors should use acetylated tubulin stain to assess whether axon number and/or organization is impaired in these animals.

We repeated all experiments and found that the IOP-treated animals for 7 days did not consistently show morphological defects. However, to attend the reviewer’s concern, we searched for an antibody to label acetylated tubulin. We finally decided against these experiments since tubulin is a very dynamic structure and we could not find a label that would depict an impairment on axonal organization or number.

MINOR:

Line 67: Myelin is not present in all vertebrates. The sentence should be clarified to state that myelination is the last major event to occur in all vertebrates for which axons are myelinated.

Corrected.

Lines 77-79: Mbp is also robustly expressed in Schwann cells. The major difference between central and peripheral myelin is the presence of Plp in oligodendrocytes and Mpz in Schwann cells.

According to several authors, mpb, mpz and plp1 are distributed in both the central and the peripheral nervous systems. We have corrected the text and included new references to support this notion (Brösamle C, Halpern ME. Characterization of myelination in the developing zebrafish. Glia. 2002 Jul;39(1):47-57. doi: 10.1002/glia.10088. PMID: 12112375).

Lines 338-339: Olig2 is not specific to oligodendrocyte precursors. It is also a primary marker of motor neurons (PMID: 12167410), which complicates the interpretation of the qPCR results as noted above.

Corrected.

Statistics: Why were medians compared rather than means?

While the graph type used (Box and Whiskers) for Figures 1, 4-6 and Supplementary Figure 2 depict medians in the middle of the box, the ANOVA analysis coupled with Tukey test always compares the means. We just used the Box and Whiskers graph type to better show the dispersion of the data. Although we could change the graph type if the reviewer insists, the statistical analysis results remain the same.

Reviewer #2:

1) Rationale for the parts of the nervous system is not clear.

We selected regions that included the forebrain, midbrain and hindbrain to have a more integrative picture of CNS myelination. We have now specified the location within the nervous system of the anatomical structures that were analyzed in this study (lines 32, 173-174).

2) Even if IOP is known to affect deiodinase activity and thyroid hormone synthesis , an embryonic treatment with IOP in zebrafish is not well linked with hypothyroidism. As hypothyroidism is not fully demonstrated, some conclusions are not supported by the data presented. Would it be possible to better show the hypothyroidism? Can T3 and T4 measurements be performed on embryos (whole or tissue) before and after IOP treatment to support the fundamental hypothesis of hypothyroidism? If not, can a T4 antibody be used? Or study of the thyroid gland? pituitary (TSH) or thyroid growth related genes?

In the present study, we aimed to reduce T3 bioavailability, rather than to induce a classical hypothyroid condition. While embryonic T4 is from maternal origin, most of the embryonic T3 is derived from T4 to T3 conversion. Impairing this conversion with IOP is the only available method to reduce T3 signaling at very early stages, before the embryonic thyroid gland is active. While measuring T4 would not offer proof of a T3 deficiency, measuring T3 would; however, very sensitive methods would be needed to indeed show this partial T3 reduction. To attend the reviewer’s concerns, we instead: 1) set up a protocol to stain and analyze larval cartilage, which is known to reflect the thyroidal status in mammals and fish. Indeed, we observed malformations in Meckel´s cartilage, revealed by Alcian blue staining (Figure 1A); 2) we measured additional TH-linked genes like thsb, ttr and gh. For these and all the genes selected to measure the thyroidal status, we designed new oligos improving their qPCR amplification efficiency. All experiments were repeated, increasing the total number of individuals analyzed per experiment. With these new conditions, we observed a clear TH regulatory pattern for both, dio2, dio3 and tshb (Figure 1B), supporting the lower T3 availability in the larvae. We were unable to detect changes in ttr and gh, suggesting that these genes are not as sensitive to T3 fluctuations, further supporting that with IOP treatment we induced a rather mild T3 deficiency.

3) The article is mostly descriptive and lacks a functional part to make the data fully usable and understandable.

To address this concern, we included two experiments to gain some insights regarding a possible delay in oligodendrocyte development due to T3 deficiency: 1) a follow-up on myelin formation using live mbp:egfp larvae at the onset of myelination and once myelin formation is ongoing; 2) immunostaining using an OPC marker to analyze if OPCs showed a developmental delay with T3 deficiency or if fewer OPCs transitioned to myelinating OLs (lines 318-330 and 398-406).

4) Statistics: with n=3 for the gene exppressions for exemple. A comparison of more than two groups by mann whitney is not recommanded (ANOVA should be used but I don’t know if n=3 is enough for statistical power).

We thank the reviewer for noting the errors. As mentioned previously, we have repeated all statistical analyses with the suitable statistical tests, running ANOVAs in the experiments where more than 2 groups were compared. Also, the n value in each experiment was increased for a more robust analysis.

Genes are quantified and related to a total amount of RNA. However, the actin B1 gene is in the list of primers used. It is not clear whether this housekeeping gene has been used for relative quantification or not.

We have extended the section on the qPCR methodology for clarity in the new version of the manuscript (lines 137-145). We explain how absolute, not relative gene quantification is performed and the use of two reference genes as correction factors.

The authors assume that the decrease in dio3 is a sign of hypothyroidism but the increased expression at 3dpf of thrb, mpa at 7 days and the decrease in thraa and thrab do not support this hypothesis. It is also debatable whether these measurements are made on whole organisms. Only an overall net effect is therefore observed and correlations with tissue or even cellular events in the nervous system are hazardous, given that opposite cellular regulations can be observed, particularly during development. A discussion on tissue and cellular effects of thyroid hormone would be therefore interesting

As mentioned in the response of point 2, we have included other criteria to determine reduced T3 availability signs in the new version of the manuscript. We have also included a sentence (lines 361-364) to include the arguments raised. However, we decided against discussing on tissue and cellular effects of thyroid hormones in the developing nervous system of zebrafish, given that we do not have detailed results in the present study and that available published information is scarce.

It is quite surprising to see that the T3 treatment alone has not been carried out on gene quantification and it is not described whether the 50/60 embryos x3 come from 3 independent experiments or if they are from the same experiment.

In the newly performed experiments, we have included results from gene quantification of T3- (Figure 1) and IOP + T3-treated groups (Figures 1 and 4). We have also clarified that each pool represents an independent experiment (lines 582 and 615).

There is no functional experience on the consequences of a delay in myelination (movement for example) and rescue that the T3 treatment could bring. Furthermore, cross sections show a lack of effect of IOP treatment on myelinated axons. How then can this apparent contradiction be explained with the drop in myelin density observed with Black gold II marking? What are the effects of T3 and IOP+T3 treatment on these parameters?

Indeed, cross sections did not show a decrease in axonal diameter. In order to confirm these results, we repeated the experiment in both, whole-mount preparations and cross-sections. The Black-Gold II staining was improved, resulting in images where myelin is more clearly observed; however, when we performed the Black-Gold II staining in cross-section, images were not optimal to accurately measure myelin content because sectioning harms the integrity of the tissue. Since the new whole-mount images depict myelin content quite unequivocally, we decided to just include these in the new version of the manuscript. For this reason, we have eliminated the Figure 3 of the original version of the manuscript and have included new images in Figures 2 and 3. In them, we observe a clear decrement in myelin content after IOP treatment at 3 and 7 dpf. However, this decrement is not reflected in movement. In our hands, we found no differences between control and IOP-treated 7dpf zebrafish larvae in natural swimming behavior using single visual inspection and through a free software analysis (Kinovea, ToxTrac). The analysis of the effects of T3 and IOP+T3 was not possible because 7 dpf larvae died during the experimental treatment, as mentioned in the new version of the manuscript. We have included this information in lines 353-356.

Fig 6/ Video Suppl 4: the term positive control is not appropriate (solvant control). In the video the size is not the same than the others 3D reconstructions.

A scale bar should be present in all sections/pictures/video

We have corrected the video.

Submitted filename: Answers to Reviewers.docx

Click here for additional data file.

16 Jun 2021

PONE-D-20-33696R1

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination

PLOS ONE

Dear Dr. Orozco,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your revised manuscript has been reviewed by the same two reviewers. Both reviewers mentioned that your manuscript has been improved but were not fully satisfied with the revision. You will have to carefully consider their recommendations and re-revise your manuscript accordingly. At this stage, I cannot guarantee that your re-revised manuscript will be found acceptable for publication in Plos One.

Please submit your revised manuscript by Jul 31 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

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Hubert Vaudry

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this revised version of “Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination,” Farías-Serratos et al. expand their analysis of the role of thyroid signaling in oligodendrocyte myelination. The implications of this study are interesting and the manuscript is well written, and the additional experiments (assessment of OL development) have added support to their initial claims. I have several concerns that need to be addressed prior to publication, mostly related to statistical rigor.

1. For the new Alcian blue stains (Figure 1A-B), the authors should show labeling and quantification of the cartilage malformation. To the general reader, this difference is not immediately apparent.

2. For all graphs, individual data points she be plotted within the bar graph or box/whisker plot to increase transparency of reporting.

3. The control images for CeP and D+DT+TeO in Figure 2 in the resubmission are not nearly as clear as Figure 2 in the original submission. Please provide representative images with less background stain.

MINOR:

1. Line 68: Myelination in mammals starts in the late stages of embryonic development, not during early stages of development.

2. Line 97-100. Need a citation or else need to state that this is the hypothesis.

3. Line 101 typo. Should read “post-fertilization in teleost”

4. Line 104 typo. Should read “later of larval”

5. Lines 126-127 are redundant with lines 121-122.

6. Lines 284/285, it would be helpful to have these structures defined for the first time here rather than in the methods.

7. Figure 5: need to label the transgene used and the developmental stage.

Reviewer #2: The review of the paper by Farias-Serratos et al on the effect of iopanoic acid on myelin was extensively addressed by the authors.

The quantitative PCRs are now clearly shown and appropiately detailed. The effects are unambiguous. The effects on tsh suggest an IOP-induced decrease in thyroid hormone and a T3-induced increase at 3dpf.

Black gold staining of myelin at 3 days and 7 days showed a decrease in myelin observed at 3 days and still visible at 7 days.

The results at 3 days are not always very convincing since only developmental delay could explain the absence of myelin in the fin . I personally find it very difficult to see a difference between BGII stained animals , without the fibers drawn in red in figures 2 and 3.

IHC with NG2 on larvae in dorsal view seem diffuse and the question of specificity arises. The authors also added experiments using the mbp:eGFP transgenic line. It would have been useful to confirm the data in Figure 3 with this model (given the paper by Jung et al 2010 and the specificity observed in the fin but not necessarily in the brain). Moreover, the signal seems very diffuse. Finally the structures, especially the telencephalon are clearly different when comparing IOP, CTRL and T3 and here again a growth delay explaining the absence of myelin cannot be ruled out.

Overall I recognize the efforts that the authors have made and the quality achieved for the transcriptomic data is very satisfactory. The article reads very well and the reviewers' modifications were generally taken into consideration. There remains this point about the brain structures, which are different (and differently myelinated) depending on the stage of development and/or treatments.

line 227 : This is not true that IOP and T3 treatment always induce gene expression going to opposite ways. For example, for dio2, they go the same sense. Actually this statement is only valid for dio3 and tshb expression.

Also in fig 4 it seems quite weard that T3+IOP is not more fluorescent than CTRL given the very high fluorescent signal in TeO.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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9 Jul 2021

We thank the reviewers for their careful revision of our manuscript. As suggested, all Figures were modified to include individual data points and/or to provide with neater and clearer representative images. Also, attending the comments raised by Reviewer #2 regarding the possibility that the absence of myelin could be related to a developmental delay due to the IOP treatment, we measured control and IOP-treated larvae at 3 and 7 dpf. As discussed in the point-by-point rebuttal, we observed that lower T3 bioavailability does impair correct myelination and that a delay on growth is not evident until 7 dpf.

Submitted filename: R2. Answers to Reviewers.docx

Click here for additional data file.

3 Aug 2021

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination

PONE-D-20-33696R2

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #2: Partly

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Reviewer #1: (No Response)

Reviewer #2: I have seen this artilce for the third time. The authors changed the figures, again .

I feel that indivual plotting is a very clear way to present data. I am still not convinced by the BGII staining especially in the forebrain where it shoudl not be seen myelination at 3dpf, according to the literature and to what authors mention in the introduction. I regret the authors did not acceed to my query of using trangenic animals. Even though I think it could have stregnthen the article I do not want to spend more time on this. Therefore I have last recommandations authors may use before publication.

IOP does not only block T4 to T3 conversion (as stated in the abstract) but also T3 inactivation. Even though I understand this is the activity the authors focused on, the IOP blocking IRD activity should be mentioned in the introduction (especially when presenting IOP+T3 results).

Line 276-277 ; treatment in 3 dpf larvae induced dio3 and tshb gene expression

Line 277 changes in the opposite way as compared with IOP-treated larvae. _The way the sentence is written a bit missleading cause tshb is decreased by T3.

Black gold staining :

To state the there is a partial rescue column to column comparison should be done between IOP and IOP+T3 groups.

There are opposite results showed by black gold staining (no T3 effects vs strong effects on qpcr). IOP+ T3 treatment reveal that T3 by itself has no effect in contrast to what could be seen at molecular level. This is partially discussed (maximum reached in CTRL and T3 but T3 accelerates the process), however it seems weard that T3 by itself has no global effect (BGII Staining or GFP)

Line 385-386 : plp1b is not seen as significatly down regulated in figure 4

Line 391-193 : This is an interesting suggestion that sox10 reveals uncomitted myelinated cells cells but how explaining IOP+T3 effect? In my opininon IOP+T3 is a super T3 effect ( no degradation of a part of T3 so more T3 available). However this is contradictory with mbp egFP results where you see a T3 reversal IOP induced effect. This should be discussed ? (following paragraph in the discussion).

Line 407 : If T3 accelerates myelination, does a significant difference be seen in mbp :egpf larvae ? as quantification is done a 3dpf ?

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Reviewer #1: No

Reviewer #2: No

6 Aug 2021

PONE-D-20-33696R2

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination

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