Effects of sim-μg on cartilage and bone formation: 5 days in clinorotation versus controls

Larvae of 5 dpf were maintained in clinorotation for another 5 days. At the end (10 dpf), controls and sim-μg-exposed larvae were stained with Alcian blue to observe the cartilage and Alizarin red to visualize calcium deposition by osteogenic cells (see Figure 1a,b). The cartilage structures are well-formed, complete and morphologically similar to the respective controls (Figure 1c,d). In contrast, bone formation was clearly decreased in the larvae submitted to clinorotation relative to their controls (Figure 1e,f) and several bone structures, such as anguloarticular, branchiostegal ray2, ceratohyal, and dentary are absent.

Figure 1 Effects in 10 dpf zebrafish larvae after 5 days sim-μg in clinorotation (a) Schematic representation of the different cranial bone elements revealed by alizarin red staining in 9–10 dpf zebrafish larvae and (b) the landmarks used for morphometric analysis. The landmarks used in morphometric analysis are anguloarticular (aa), anterior (an), branchiostegal ray1 (br1), entopterygoid (en), maxilla (m), notochord (n), opercle (o), and parasphenoid (p). Note that the parasphenoid is a triangular bone defined by its anterior summit (a) and two posterior summits (b,c). Additional bone elements that were evaluated for extent of ossification are: branchiostegal ray2 (br2), cleithrum (c), ceratobranchial 5 (cb), ceratohyal (ch), dentary (d), and hyomandibular (hm) (from ref. 9). (c,d) Alcian blue staining of cartilage. (e,f) Alizarin red staining of bone structures. Compared with controls (C), no effect was observed on cartilage development after 5 days clinorotation (D). In contrast, relative to control (E), a clear decrease of bone formation was seen after 5 days in clinostat (F). Scale bars=250 μm. (g) Morphometry. A significant increase of the distance between the anterior part of the larvae and the parasphenoid summit is observed. The distances are measured in pixels. Mean±s.d. and t-test analysis were calculated for each measure on at least 20 individuals. (h) The area covered by the parasphenoid is decreased on clinostat exposure. (i) Extent of bone formation: Bone development is classified for each element into different categories: Absent (no structure present; red), early ossification (beginning of the bone ossification; yellow), advanced ossification (the structure is present and already developed as the control; green), and over ossification (the structure is more developed compared with the control; purple). Cumulated frequencies in % are represented for each element. As no significant difference was observed for paired structures between left and right (up and down), their scores have been combined. Statistical analysis was performed by X2 of Pearson and a logistic regression. (i) Cumulated frequency after 5 days in clinostat. (j) To obtain global scores, the scores attributed to each element were added up for each individual larva. Mean±s.d. and t-test analysis was obtained on at least 20 individuals. **P<0.005 and ***P<0.001. dpf, days post fertilization. Full size image

The extent of bone formation was analyzed more precisely by using qualitative and quantitative descriptions of the images of the stained larvae by applying two approaches.9

The morphometric approach

Each image was manually annotated by defining specific landmarks indicating the positions of the different skeletal structures. Symmetric structures, such as the maxilla (m), entopterygoid (en), branchiostegal ray (br), or opercle (o) were distinguished by the suffix “up” or “down” (see Figure 1a,b). The software then calculates all the distances between the selected landmarks to obtain a morphometric description of the head skeleton. In the cartilage skeleton, larvae subjected to clinorotation did not reveal any significant modifications (data not shown). In contrast, larvae stained with alizarin red revealed a clearly increased distance of the parasphenoid summit (pa) and the anterior (an) part of the larvae (Figure 1g, Supplementary Table S2), probably due to the significant decrease of the parasphenoid (p) area (Figure 1h, Supplementary Table S2). Note that not all of the measures were possible due to the absence of several structures in >60% larvae such as the anguloarticular (aa) and ceratohyal (ch) (see also below).

The staining intensity evaluation approach

According to its developmental status (absent, early, advanced, or over ossification), a score, from 0 for absent to 3 for advanced ossification, is given to each structure. The frequency distribution of these scores reveals that exposure to clinorotation lead to a significant decrease in ossification of all the structures (Figure 1i, Supplementary Table S3). The branchiostegal ray1 and the entopterygoid structures were absent in ~25 % of the treated larvae, while they presented advanced ossification in 100% of the control larvae (Figure 1i). The anguloarticular and the ceratohyal were absent in about 60% of the treated larvae, compared with 25% or 15%, respectively, in the controls. Absence of the branchiostegal ray2 switched from 25% in controls to about 80% after clinorotation. For all structures, the frequency of larvae presenting advanced ossification significantly decreased on clinorotation. Assigning a global score for all structures within each larva confirmed that 5 days of clinorotation treatment produced a significantly decreased ossification (Figure 1j).

Sim-μg and stress in larvae

Stress can induce bone loss;10–13 therefore we decided to evaluate the stress status of 6 dpf larvae after 1-day exposure to sim-μg. To this end, we determined the whole body cortisol levels in 15 larvae directly after sacrifice. This analysis has been performed through subsequent adjustments:

(i) To collect and kill the larvae, we compared two different methods, shortly defined as low-tricaine and high-tricaine. Low-tricaine: to cause a rapid anesthesia followed after 2–3 min by death by adding tricaine to a final concentration of 0.04 g/l;14 high-tricaine: to cause immediate death by collecting larvae in a reduced volume of E3 medium and adding 1.6 g/l of tricaine. Since we observed significantly higher cortisol levels in larvae killed with low-tricaine (Figure 2a), probably due to the acute stress induced by anesthesia, we applied the high-tricaine method in all experiments.

Figure 2 Evaluation of stress by cortisol assay. (a) Negative and positive controls. Low-tricaine dose, agitation, and salt water significantly increase the cortisol level compared with high-tricaine. (b) No change in cortisol level in clinostat compared with their control. ***P<0.001 Full size image

(ii) To induce acute stress in zebrafish larvae, we performed positive control experiments by using two different known procedures: the first consists in intense agitation for 30 s in 5 ml medium followed by a 5-min resting period15 before sacrifice at high-tricaine, while the second exposes the larvae to a 1.75 g/l NaCl solution for 5 min before leaving them for 5 min recovery in E3 (ref. 16) and then sacrifice. As expected, both stress conditions lead to a significant increase in cortisol levels, which interestingly were similar to the levels observed under low-tricaine conditions (Figure 2a).

(iii) Finally, careful measurements of the cortisol levels indicated no differences between the larvae subjected to clinorotation for 1 day as compared with their respective controls (Figure 2b). These results demonstrate that the larvae placed into this microgravity simulator are not stressed compared with their respective controls. Thus, any modification observed is most likely related to the effect of sim-μg and not stress.

Effects of sim-μg on gene expression: 1 day clinorotation versus controls

To obtain a global view of the physiological changes caused by sim-μg, we performed a microarray whole genome expression analysis. We compared 6 dpf control larvae with larvae growing in clinorotation for 1 day, i.e., from 5 dpf to 6 dpf, to capture early-regulatory events rather than secondary regulations, leading ultimately to the observed modulations of bone formation at 10 dpf. Four independent experiments were carried out, using 60 larvae for each experimental condition. Due to the small volume available in the rotating tubes in clinorotation, only 15 larvae were run in parallel in three tubes; thus each control or rotated sample consisted of a pool from 4 different experiments to reach a sample size of 60 larvae. Total RNA was extracted from 6 dpf control larvae and larvae that experienced sim-μg, reverse transcribed into complementary DNA, and used for gene expression microarray analysis.

A list of genes affected more than 1.4-fold (|log2 fold change|>0.49) was extracted and introduced into the Ingenuity Pathway Analysis software (IPA) for further analysis. In total, 208 genes were significantly affected in the clinorotation experiment, of which 66 genes were annotated in IPA. The full list of these genes is given in Supplementary Table S4. Validation by reverse transcription-quantitative PCR of five genes selected from the list demonstrated the reliability of the microarray data (Figure 3a).

Figure 3 Genes whose expression is affected by clinorotation for 24 h starting at 5 dpf. (a) Fold change values on clinorotation treatment for selected genes. The fold change after clinorotation between 5 and 6 dpf is given as deduced from the microarray data and the RT–qPCR confirmation experiments. For microarray data, only significant fold-change values are shown, while for RT–qPCR bold type indicates significant changes in expression (P<0.05). (b) Diseases and Biological functions affected by clinorotation. IPA analysis of the list of genes affected at 6 dpf after 1-day clinorotation compared with controls. The columns represent the −log(P-value) for significance that the list of genes affects the indicated biological function. (c) Upstream regulators, as suggested by the lists of genes affected at 6 dpf after 1-day clinorotation as compared with the corresponding controls. Potential upstream regulators known to modify expression of genes in the gene list were identified, the numbers given are the z-scores indicating significance of the upstream regulator, negative z-scores correspond to predicted decreased activity, while positive z-scores correspond to a predicted increased activity of the proposed regulator. (d) Genes connected to the upstream regulator CREB1 as predicted by Ingenuity Pathway Analysis. CREB1 activity is predicted to be downregulated in clinorotation condition (blue color). Blue or orange lines indicate, respectively, inhibition or activation of expression consistent with the prediction, gray arrow indicates that no information is available (inconsistent findings would be in yellow). Arrows indicate an interaction activating, while stop-lines indicate an interaction inhibiting expression of the target gene. Red overlay color indicates increased gene expression, while green overlay indicates decreased gene expression in the corresponding experiment. Full size image

In general, it appears that the number of significantly affected genes is relatively low, indicating that sim-μg has no major immediate impact on general physiology. The most highly affected gene in clinorotation (Supplementary Table S4) is AXIN2 (log2 fold −3.48), indicating a downregulation of the Wnt pathway, while the most highly upregulated gene is HES1 that is involved in NOTCH signaling.17 Other affected genes are IGF2R, a component of the insulin-like pathway, or the ryanodin receptor RYR2. Affected transcriptional regulator genes are also represented, such as E2F2, FOSB, or the bone-specific factor HOXB9.

IPA was used to identify the biological functions and regulatory pathways that were affected by sim-μg. Among the ‘canonical pathways’ affected (Supplementary Table S5), the retinoid X receptor RXR is prominent in its common role for FXR/RXR, VDR/RXR, and FXR/RXR signaling, while other pathways regulated by IL-6, Notch, VDR, Hif1ß, LPS/IL-1, and Notch were also affected. Classification of the list of affected genes according to their involvement in specific ‘Disease and Biological Functions’ revealed their role in morphology, size, and resorption of bone, as well as the quantity of osteoclasts, bone, and blood cells. When considering the more generic ‘Disease and Biological Function’ terms according to their relevance (Figure 3b), ‘Connective Tissue Disorders’, Skeletal and Muscular Disorders, and ‘Connective Tissue Development and Function’ ranked on position 2, 5, and 11, respectively. Finally, we analyzed the data set for putative upstream regulators and the predicted change in activity of these regulators (Figure 3c) and we identified CREB1 and CREM as putative affected regulators, but also TCR, IL-12, IL-6, and the bone-specific transcription factor RUNX2. The observed changes in gene expression are indeed consistent with a decrease in CREB1 activity in clinorotation (Figure 3d).

One important aspect of our study is the fact that we investigated gene expression using mRNA from the entire larvae. We first filtered the gene list according to their involvement in musculoskeletal or cardiovascular function or disease (Supplementary Table S7), we identified 8 genes common to both systems, 9 genes related to the musculoskeletal function, and 10 genes more specific to the cardiovascular system. When we focused on individual organ systems by filtering the affected gene set against available databases of genes involved in specific functions (gene ontology annotations of human or mouse gene orthologs using the IPA knowledge base), networks of regulatory interactions could be constructed for each system. Specific to bone, a small pathway centered around FGF2 (increased expression) and including FOSB, GAB2, and E2F2 (decreased expression) was identified. In muscle, FOSB is absent, but FGF2 is additionally connected to MCL1 through GAB2. Further, the increased expression of CYP19A1, required for estrogen synthesis, and its effect on lipoprotein metabolism was also pointed out. In the cardiovascular system, all these genes and pathways are represented