Muscle defects due to perturbed somite segmentation contribute to late adult scoliosis

Somite segmentation mutants present vertebral defects with variable levels of scoliosis

As we have previously shown (Lleras et al., 2018) zebrafish mutants for her1^-/-; her7 ^-/- and her1^-/-; her7^-/-; tbx6^-/- present mild defects in the segmentation of the axial skeleton (fusion and hemivertebrae) with a 100% incidence. This is also true for 80% of fss (tbx6^-/-) mutants [23]. Later observation of these three different mutant genotypes revealed that during aging they develop different degrees of scoliosis. In order to record this phenomenon properly, a time-lapse series of photographs were taken of eight mutant and wild type control individuals from each genotype from 6 weeks post fertilization until one year (Figure 2A). Up to the age of 6 weeks there was no phenotypically visible scoliosis. The angle of scoliosis (the angle of deviation from the body axis) was measured from images as described in Materials and Methods (Figure 2C and 2D). In all three zebrafish mutants analysed the first signs of scoliosis could be detected at 3 months post fertilization (Figure 2B–2D).

Figure 2. Clock segmentation mutants develop adult scoliosis. (A) Representative time lapse images of individuals from each genotype over the period from 6 weeks to 12 months, allowing to track the development of scoliosis (arrow). Mutant individuals have already mild signs of scoliosis at 3 months, while wild type exhibit the first indication of deviation from the body axis at 6 months. In the her1^-/-; her7^-/- individual at 3 and 6 months is an exemplary depiction of how scoliosis measurements were carried out. (B) Dorsal view of the different genotypes, showing an S body shape characteristic for scoliosis. (C) Graphical representation of the percentage of fish developing scoliosis over time, reaching 100% in the triple mutants and 83% in the tbx6^-/- at the end point. In the wild type and in the her1^-/-; her7^-/- mutants only 38% presented scoliosis. (D) Measurements of axis angles in different individuals at different time points during virtual time-lapse. Only the individuals with an angle of deviation from the body axis are shown in the graph, none bended individuals have an angle of zero. Different line colours represent individual fish. Between 9 and 12 months, two wild type fish (1 with scoliosis and 1 without scoliosis), one her1^-/-; her7^-/- (with scoliosis) and two tbx6^-/- (both with scoliosis) had to be sacrificed. After the individual was removed, it was still counted as bended or normal in the 12-month quantification. Note: the angle can decrease or increase depending on how the angle of deviation from the body axis develops over time in the individual. The ruler in section A serves as a scale bar, the space between two successive lines marks one millimetre. The scale bar in section B represents 1 cm.

In order to show that onset of scoliosis occurred at a similar rate in the respective genotypes we evaluated third generation offspring obtained from crosses of those first generation fish used in the initial time lapse. At three months post fertilization 11 out of 35 (31.4%) tbx6^-/- third generation animals developed scoliosis, similar to first generation animals 3 out of 8 (38%) evaluated at a similar age (Figure 2C and 2D). At the same time point, 8 out of 38 (21%) her1^-/-; her7^-/-; tbx6^-/- third generation animals presented scoliosis in contrast to first generation animals 5 out of 8 (63%). By the end of the experiment (12 months post fertilization) all her1^-/-; her7^-/-; tbx6^-/- first generation animals (8/8) had developed scoliosis. Similarly, in the third generation, all animals (7/7) her1^-/-; her7^-/-; tbx6^-/- animals developed scoliosis at 14 months post fertilization (Figure 2D). By 12 months 88% (6/7) first generation tbx6^-/- animals had developed scoliosis (Figure 2C and 2D); this corresponds exactly to the proportion of individuals from these two mutant populations that present vertebrae defects (see Lleras- Forero et al., 2018). Scoliosis in the her1^-/-; her7^-/-; tbx6^-/- mutant background was much more severe than in her1^-/-; her7^-/-; or tbx6^-/- mutants (Figure 2A, 2B and scoliosis angles in Figure 2D). Despite all her1^-/-; her7^-/- mutants showing vertebral fusions and hemivertebrae, only 5 out of 25 (20%) of the third generation and 38% (3/8) of the time lapse individuals developed visible scoliosis at 12 months post fertilization. All of the her1^-/-; her7^-/- mutants had vertebral fusions and hemivertebrae in more than one position within the axial skeleton. This value was the same as the wild type scoliosis recurrence. In wild type individuals, scoliosis was first detected at 5 months post fertilization in 1 out of 22 fish (4.5%) and at 6 months post fertilization in 1 out of 8 (12%). At the end of the analyzed period (1 year) 38% (3/8) of wild type animals had developed scoliosis. These individuals had no vertebral defects. Presentation of scoliosis therefore did not correlate with axial skeletal phenotypes in wild type animals. An additional observation, showed that 64% of the females (9/14 that developed scoliosis during the whole experiment) had the first measurable sign of scoliosis at 3 months post fertilization compared to 0 % of males (Figure 2D).

Muscle volume is altered in somite segment mutants in regions that correspond to spinal curvature

In order to establish if the volume of the muscles in adults carrying mutations affecting somite segmentation was different to wild type animals, all individuals were stained with contrast medium and imaged by micro CT at 12 months of age. The volume of the left and right hypaxial and epaxial muscle of each individual was measured at four positions relative to body landmarks. These landmarks were (1) beginning of the pectoral fin, (2) end of pectoral fin, (3) pelvic fin and (4) anal fin. The first and second positions were selected as the majority of the scolioses were observed in the fish at these levels. The musculature of the fish changes in volume significantly at different anterio-posterior positions, such that anterior myomeres are larger than those in posterior region. By standardizing the locations of all measurements of muscle volume for each animal, we could compare absolute muscle volumes between animals. This allowed us to ensure that any differences in muscle volume were not due to inherent differences in muscle volume along the trunk or due to differences in animal size (Figure 3A–3E). A two-tailed student´s t-test analysis showed that all three mutants have a smaller muscle volume at the beginning and end of the pectoral fin compared to wild type animals (Figure 3F and 3G). In tbx6^-/- mutants, the muscle volume at the pelvic fin and the anal fin was also statistically different from wild type (Figure 3H and 3I). her1^-/-; her7^-/- individuals that presented scoliosis during the course of the experiment (Figure 3: red bars in bar chart are scoliotic individuals; each bar is a single individual) tended to have the same muscle volume as their phenotypically normal her1^-/-; her7^-/- mutant siblings (Figure 3: blue bars, non- scoliotic individuals; each bar is a single individual). In contrast tbx6^-/- mutants with scoliosis showed a smaller muscle volume at all anterior positions compared to non-scoliotic tbx6^/- mutant animals. This observation suggests that in tbx6 ^-/- mutants scoliosis correlates with lower muscle volume. No difference was seen between the muscle volume of male and female individuals.

Figure 3. Muscle volume is affected in mutants at adult stages. (A–D) Representative micro CT images showing the dorsal view of fish used for the segmentation of muscles into the left and right side at the four key areas (indicated by the colored regions) in the 4 different groups: (A) wild type, (B) tbx6^-/- (C) her1^-/-;her7^-/-, and (D) her1^-/-;her7^-/-;tbx6^-/-. (E) Lateral view of reconstructed surface generation of the individual muscle from the WT. (F–I) graphical results of the volume measurements for every area. (F) Muscle volume at the beginning of the pectoral fin. (G) Muscle volume at the end of the pectoral fin. (H) Muscle volume at pelvic fin level. (I) Muscle volume at anal fin level. An asterisk denotes a statistically significant difference between wild type and mutant groups (two tailed significant test P=0,05). Individuals that presented scoliosis during the course of the experiment (represented with red bars) tend to have the same muscle volume as their phenotypically normal siblings (represented with blue bars). The three mutants analyzed have less muscle volume at the first two anterior positions. The muscle of 27 out of 32 individuals could be analyzed to 12 months, because two wild type fish (1 with scoliosis and 1 without scoliosis), one her1^-/-; her7^-/- individual (with scoliosis) and two tbx6^-/- individuals (both with scoliosis) had to be euthanized between 9 months and one year. After the individual was removed, it was not stained for muscle analysis and therefore will not appear in the graph.

Three-way correlation analysis was performed in order to establish if the length of the fish had an effect on the muscle volume or the angle of deviation from the body axis. It was found that the length of the fish has a negligible correlation with the angle of deviation of the body axis (-0, 26). There is a low positive correlation between muscle volume and fish length (0,47) and a low negative correlation between muscle volume and angle of deviation (-0,39). An interpretation of these correlation scores is that severity of scoliosis correlates with a lower muscle mass but is independent of the length of the fish.

Further analysis of orthogonal sections of the muscle of animals from Micro CT imaging showed that the muscle fibers in the mutants join the vertebrae in a disorganized manner and borders between the myotomes were not distinct in her1^-/-; her7^-/-; tbx6^-/- or tbx6^-/- mutants (Supplementary Figure 1). In contrast, in her1^-/-; her7^-/- mutants, the borders between the individual myotomes were clear but completely disorganized and did not align between the dorsal and ventral sides (Supplementary Figure 1). In addition, animals for all three mutant genotypes possessed cavities in the muscles (arrowheads, Supplementary Figure 1), that were not seen in wild type individuals. Haematoxylin and Eosin staining of transverse sections of wild type and mutant animals clearly revealed disorganization of muscle fibers (Supplementary Figures 1 and 2). In all three mutants analysed, the muscle fibers appeared shorter (data not quantified) and lost their characteristic parallel organization in the myoseptum and the normal transverse morphology at the dorsal and ventral sides. In addition, in all three mutant groups, the shape of the fibers appeared to be variable (Supplementary Figure 2).

Fast muscle area and fiber size is affected at embryonic stages in her1^-/-; her7^-/- mutants

Myotome boundaries and the axial skeleton were characterised at 15 days post-fertilization (dpf) (Supplementary Figure 3) to determine whether segmentation defects persist and whether this correlates with axial skeletal defects (axial skeletal defects are indicated by arrows). In all mutants studied, the myotome boundaries were disorganized compared to wild type controls at 15 dpf. Furthermore, in the tbx6^-/- and the her1^-/-; her7^-/-; tbx6^-/- mutants, there was a decrease in the number of cells expressing the myotome boundary marker (gSAIzGFFM1954A) (Supplementary Figure 3). Therefore, myotome boundary defects persist throughout development in the whole zebrafish body axis and not only where axial skeletal defects are present.

To investigate further the early muscle phenotypes of these mutants, 32 hours post fertilization (hpf) embryos were analyzed. In all mutants the fast (Phalloidin positive) and slow (F59 positive) muscle fibers lose their metameric organization (Figure 4A–4L). In addition, in the tbx6^-/- and the triple her1^-/-; her7^-/-; tbx6^-/- mutants cavities can be seen in the fast muscle (Figure 4B´, 4D´). These can vary in size and number (Figure 4L´). In tbx6^-/- mutants, cavities were found in 15 sections out of 20 sections analyzed. 30 cavities (between 1 and 5 per section) could be measured with an average size of 1.105μm ± 0.66. In her1^-/-; her7^-/-; tbx6^-/- cavities were found in 18 out of 20 optical sections, 40 cavities (between 1 and 5 per section) were measured with an average size of 1.407μm ± 0.93. In order to determine whether the cavities contained cells that do not express muscle fiber markers, whole mount DAPI staining was performed. In all her1^-/-; her7^-/-; tbx6^-/- and tbx6^-/- embryos imaged at 32hpf (n=8 for each genotype), the cavities did not contain any cells (Supplementary Figure 4). In addition, the her1^-/-; her7^-/-; tbx6^-/- mutants present an agglomeration of MF20-labeled fibers around the vacuolated regions (Figure 4L´). The slow fibers in the tbx6^-/- and the triple her1^-/-; her7^-/-; tbx6^-/- mutants appeared to be fused and thicker than in the wild type. In several of these animals there were regions of the slow muscle in which no fibers were present (Figure 4H´) or the slow fibers appeared to invade the inner region containing fast muscle fibers (Figure 4F´) as previously described in fss mutants [24].

Figure 4. Fast and slow muscle fibers are affected in mutants at embryonic stages. (A–D) Phalloidin staining for fast muscle fibers in all mutants show loss of the characteristic metameric structure. (A) In addition, the transverse plane sections at the cloacal level in tbx6^-/- and her1^-/-; her7^-/-; tbx6^-/- display hollow cavities where no muscle fibers were present (B´ and D´). F59 staining for slow muscle fibers (E–H) also demonstrates loss of the characteristic metameric patterning in the mutants. tbx6^-/- and her1^-/-; her7^-/-; tbx6^-/- mutants present fusions of the fibers and cavities (F, F´ and H´). The her1^-/-; her7^-/- slow fibers resemble wild type fibers in sections (G´). The marker for striated muscle, MF20 (I–L) shows the same phenotype as phalloidin and allows the visualisation of aggregates of the fibers near the areas where there are lesions in the her1^-/-; her7^-/-; tbx6^-/- mutants (arrow in L´). Quantifications of fast muscle area (M) and fiber cross-sectional area (N) at 32hpf display a statistically significant decrease (p= 0.03 and p= 0.009 respectively) of these two criteria in the her1^-/-; her7^{-/ -} mutants. In both figures, 6 points were measured. An asterisk denotes when the difference between the wild types and mutants is statistically different (p<0.05).

Quantification of the cross-sectional area of the fast muscle was performed from optical sections. This analysis showed that the her1^-/-; her7^-/- mutants have a significantly smaller region of fast muscle compared to the wild type at 32 hpf (Figure 4M). Individual fast muscle fiber cross-sectional area was also decreased at 32hpf in her1^-/-; her7^-/- mutants (Figure 4N). Fast fiber cross-sectional area is furthermore reduced in tbx6^-/- and her1^-/-; her7^-/-; tbx6^-/- mutants, but the difference is not significant when compared to wild type animals.

Animal procedures

The work was carried out at the Hubrecht Institute (the NL), according to local laws. Ethical approval was obtained through the relevant DEC committee (HI 10.1801). Standard husbandry conditions applied, according to FELASA guidelines [47]. Embryos were kept in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) at 28°C. For anesthesia, a 0.2 % solution of 3-aminobenzoic acid ethyl ester (Sigma), containing Tris buffer, pH 7, was used.

Transgenic lines and genotyping

The fss^sa38869 and her7^hu2526 mutants were provided by Jeroen den Hertog (Hubrecht Institute). The gSAIzGFFM1954A line was obtained by a gene trap method [48]. Double mutants for her1 in the her7^hu2526 background, were generated as described in Lleras-Forero et al. [23]. DNA was isolated from fin biopsies (AZ 81-02.05.40.19.044) and from embryos. Genotyping was performed as described in Lleras-Forero et al. [23].

Alizarin Red bone and Hematoxylin and Eosin staining

Hematoxylin/eosin (H&E) was performed on sagittal and transverse cryo-sections (15μm) of adult zebrafish according to standard procedures. H&E sections were photographed on a Nikon eclipse NI with a DS-Ri2 camera and 4x Plan Fluor objective. Alizarin Red staining of bone was performed as described previously [49]. Whole mount stained fish were documented on an Olympus SZX16 stereoscope with a Leica DFC450C camera.

Whole mount immunostaining

32 hpf embryos were dechorionated and fixed in 4% PFA overnight. Phalloidin (Invitrogen Alexa Fluor 546) staining was performed according to Goody, 2013 [50]. Staining with MF20 antibody (supernatant) (Developmental Studies Hybridoma Bank, university of Iowa) (1:20) was performed overnight. Embryos were first permeabilized in ice cold acetone for 20 minutes and treated with proteinase K for 30 minutes at room temperature. Secondary antibody (Dianova donkey anti mouse conjugated Cy3) was used at a concentration of 1:250. For the F59 antibody (supernatant) (Developmental Studies Hybridoma Bank, University of Iowa) (1:20), embryos were treated with 3% hydrogen peroxide for 1 hour on ice, followed by permeabilization and proteinase K treatment as described above. Samples were incubated with primary antibodies overnight. Labelling with the F59 antibody utilised amplification with the Tyramide system (PerkinElmer Inc.) by combining detection with a secondary antibody (goat anti mouse IgG/IgM HRP (Millipore)) (1:250) and the TSA ^TM plus Cyanine 3 system (PerkinElmer, Inc). For detection of nuclei DAPI was used in which embryos were fix at 32 hpf as described above and then washed twice for 10 minutes in PBS. Staining was performed with DAPI solution (Roth, final concentration of 0.2μg/μl) in Eppendorf tubes, at 4 degrees in the dark, over two nights.

Image acquisition was performed by embedding embryos in 0.8% agarose and visualising on a Leica SP8 confocal microscope using a 20X objective (N.A.= 0,75) for the whole mounts and a 40x objective (water immersion, N.A. =1,0) for the cloacal area acquisitions.

Photographic record of scoliosis

Embryos from mutant crosses were kept in E3 at 28°C until 7 dpf. They were then housed at a density of 50 embryos in a 3 Liter tank. At six weeks post fertilization, eight juvenile animals, with fully developed swim bladder, from either mutant or wildtype embryos were housed individually in the animal facility. Individuals were fed tetrahymena in combination with Gemma 75 for the first two weeks, followed by Artemia and Gemma 150 for the following two weeks. The same day each individual was anesthetized (as described above) and photographed using an Olympus SXZ16 stereomicroscope (1.5X PlanApo objective) connected to a DFC450C Leica camera. Immediately afterwards, the individual was returned to warm E3 media without anesthesia. Test subjects were returned to their specific tank in the animal facility only when they were completely awake and moving. This procedure was repeated at 3, 6, 9 and 12 months after fertilization. From 3 months onwards, a picture was taken using a Sony Xperia mobile. Sedation and photography did not take more than two minutes per animal and did not compromise survival.

Micro-CT imaging and muscle volume measurement

One-year-old fish were fixed in 4% paraformaldehyde overnight at 4 degrees; afterwards an incision was made in the abdomen and the organs were removed. Staining for soft tissue visualization with Micro CT was performed according to Descamps et al., 2014 [51] for 9 days. After staining, the samples were placed in 30% methanol for at least 24 hours. For visualization, the samples were embedded in 1% agarose inside 15 ml Falcon tubes. For Figure 2 the samples were imaged using the Bruker SKYSCAN 1272 micro-CT system. Several scans were conducted for each specimen in a batch, to ensure that the complete specimen was imaged with sufficient optical resolution. Isotropic voxel size was set to 9 mm, with 60 KeV X-ray energy, 50 W current and a 0.25 mm aluminium filter. 1501 projections were collected during a 180^° rotation, with 400 ms exposure time. Reconstructions were performed using NRecon (Version 1.7.1.0). Each individual scan overlapped its neighbouring scans in its respective batch by 200 mm, to ensure robust concatenation. For Supplementary Figure 2 the Micro CT scanning was done using the Skyscan 1176 at 65 kV, 385 μA, 1mm Al filter, 0.5° rotation steps, an image pixel size of 8.52 μm and exposure set to 1065 ms. Reconstruction of sections was carried out with NRecon v1.6.10.4.

Reconstructions were processed using ImageJ/Fiji [52]. Concatenation was performed by hand, by identifying the overlapping regions between scans and copy-pasting the batch data into the same folder. Using a custom macro (see below), each concatenated reconstruction was processed into 10-slice (90 mm) thick ‘virtual thin sections’ [53] using the “z projection” tool. This aided interpretation of muscle and increased the throughput of analyzed projections by dividing the number of effective slices per-specimen by ten. Processed stacks of virtual thin sections were then analyzed in Avizo (version 9.0; Visualisation Sciences Group). The specimen was viewed using the “Volume rendering” tool and segmented using the “Edit New Label Field” tool. For every specimen, the epaxial/hypaxial musculature was isolated and segmented in four regions (Figure 2A). Each region was divided into its right and left hemisphere, and the muscle was segmented for 10 virtual thin sections (90 mm). Volumes of every region were then calculated using the “Material statistics” tool. The statistical significance of the data was measured using a two-tailed T-test with unknown variance in Excel 2016.

Muscle area, fiber size and cavity size quantification in 32 hpf embryos

Using a Leica SP8 confocal with a 40x plan Apo objective (water immersion, N.A. =1,0) and a Z-step size of 0.27μm, an image from the cloacal region from six 32 hpf embryos (stained with phalloidin), was taken. Five slides within each image were randomly chosen and the area was measured manually using Fiji. The data point plotted in the graph is the mean of the 5 slides within one embryo.

Cross-sectional area quantification was done in the images of the cloacal region described above. Using Fiji, 5 randomly selected fibers were manually measured in the slides described above. Each point in the graph represents one embryo (25 different fibers). The statistical significance of the data was measured using a two-tailed Student’s t-test with unknown variance in Excel 2016 (Microsoft).

Cavity size in tbx6^-/- and her1^-/-; her7^-/-; tbx6^-/- were measured in the optical sections stained by Phalloidin as described above. Using Fiji, the longest vertical distance of each cavity was measured in μm. The average and standard deviation were calculated for each mutant.

Scoliosis angle measurements in photographs

In order to measure accurately if individual zebrafish had developed scoliosis and if the scoliosis progressed over time, the individual pictures from the time lapse were used. A straight line was drawn from the tip of the premaxilla, through the middle of the eye to the caudal fin. In fish lacking scoliosis or with a perturbation of the body axis the line ends exactly at the apex of the caudal fin. In a fish with a bent body axis, the line deviates from this position. The angle between the position of the line coming from the premaxilla to the new position of the apex was manually measured. In humans, the spine is considered deformed when the Cobb angle is ≥ 10 degrees [3]. The same criteria were considered for classifying the angle of deviation of the body axis for zebrafish.