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More recently, smaller fishes such as swordtail [ 37 , 40 ], medaka [ 41 — 46 ] and zebrafish [ 47 — 51 ] have attracted more attention.

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Most analyses using fishes have concentrated on the impact of altered gravity on graviperception [ 33 , 52 ], the vestibular system [ 37 , 53 , 54 ] and its involvement in motion sickness [ 38 , 55 — 57 ]. Several studies also revealed that general embryogenesis of various organisms is not affected by gravity conditions review in [ 46 , 49 , 50 , 58 ]. Here, we investigate the effect of increased gravity on the general physiology of zebrafish larvae by using a Large Diameter Centrifuge LDC [ 59 ] to study whole genome gene expression. We investigate in more detail the effects on head skeleton development and we validate our approach by studying the effects of drug treatments VitD3 and PTH known to affect bone formation.

Finally, we propose a novel approach to study the effects of microgravity by growing zebrafish in hypergravity for 5 days from 0—5dpf before returning them to normal gravity, the Reduced Gravity Paradigm, RGP [ 60 ]. The light cycle was controlled 14 h light, 10 h dark. Fish were fed twice daily with dry powder ZM fish food adapted to their age and once daily with fresh Artemia salina nauplii ZM fish food. Larvae aged less than 14 days were also fed twice daily with a live paramecia culture.

Wild type embryos were used and staged according to [ 62 ]. The day before breeding, wild-type adult male and female zebrafish were set up in several breeding tanks, separated by a clear plastic wall. After the light was turned on the next morning, walls are removed, eggs are generated by natural mating and collected from 30 minutes to 2 hours after spawning. The chemical protocol was inspired by Fleming and collaborators experiments [ 63 ]. Larvae at 5dpf were transferred into a 6 well plate Millipore containing E3 medium supplemented with the required chemical or vehicle DMSO as negative control.

The medium was changed every day at the same time. Each well contained 20 fish in 4ml. The larvae were euthanized by tricaine overdose 0. It is composed of a central axis linked to 2 perpendicular arms, each arm terminating in 2 opposing gondolas where it is possible to install an incubator containing the samples. The arms provide an 8m diameter for rotation and can provide centrifugal forces of maximum 20g. The zebrafish larvae were incubated in 20 ml E3 in a Petri dish placed in an incubator within a gondola for 3g experiments, and placed either in an incubator on the centrifuge axis axe or outside of the centrifuge for 1g controls.

In this setting, the medium represents less then 5 mm of water column and thus the 3g acceleration causes an increase in hydrostatic pressure of maximum 0. During acid-free bone structure staining with Alizarin red, bleaching was performed immediately after fixation, before the staining. The larvae are stained in a 0. Image analysis was performed on the pictures of larvae stained with Alcian blue for cartilage or Alizarin red for bone. Individual cartilage and bone elements were identified according to [ 10 , 15 , 66 — 68 ].

The program then defines the positions of all selected landmarks and computes all the distances in pixels and angles in radian of all the possibilities between two points of interest. These data were exported into an Excel file and a selection of interesting measures was conducted by performing principal component analysis on data obtained from differently treated larvae to identify invariable or redundant measures. The measures selected were: for cartilage Alcian blue : Anterior to Ethmoid plate, Anterior to Posterior, Articulation down to Articulation up, Ceratohyal ext.

A Alcian blue staining of head cartilage representing the landmarks used for morphometry. B Schematic representation of the different head cartilage elements. C Alizarin red staining of cranial bones representing the landmarks used for morphometry. D Schematic representation of the different cranial bone elements with 29 landmarks used for chemicals treatments and 15 landmarks for the 3g and the relative-hypergravity. The 15 landmarks are anguloarticular aa , anterior an , branchiostegal ray1 br1 , entopterygoid en , maxilla m , notochord n , opercle o , parasphenoid p.


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Note that the parasphenoid is a triangular bone defined by its anterior summit a and two posterior summits b,c. The 29 landmarks include the 15 named before with branchiostegal ray2 br2 , cleithrum c , ceratobranchial 5 cb , ceratohyal ch , dentary d , hyomandibular hm. E-J 10dpf zebrafish larvae after 5 days chemical treatments. E-G Alcian blue staining of cartilage.

H-J Alizarin red staining of bone. F,G no significant effect of, respectively VitD3 and PTH on cartilage development, nor on chondrocyte shape or size inlays showing close-up. I: increase of bone development after VitD3 treatment. J decrease of bone development after PTH treatment. Statistics were performed using GraphPad Prism5. A t-test was used for control versus treatment experiments, while a one way ANOVA was used for multiple comparisons.

Morphometric analysis did not inform about the extent of ossification within each larva. Thus, a systematic structure analysis was generated. Each bone structure was classified based on the progress of development into one of the four following categories: absent, early ossification, advanced ossification and over ossification.

A contingency table considered ordinal values distributed among the 4 classes from absent to over ossification or only 3 classes when one class was not present in the sample. Statistical analyses were performed using the Statistica Software version Larvae at 6dpf, after 24h treatment, were used for the RNA extraction. Purity of the amplicons was checked by melting curves at the end of each reaction. Ct values were exported from the onboard software as a text file and imported into a customized Microsoft excel spreadsheet.

Samples were run in triplicate in optically clear well plates ABgene , sealed with optical adhesive film Applied Biosystems. A good aRNA probe had a labeling efficiency of 30—50 fluorochromes every bases. The hybridization solution was prepared by adding Finally, slides were washed 3 times in room temp with 0.

Scanning was performed at a level just before saturation of several spots. For within-array and between-array normalization, print tip Loess and scale were used, respectively [ 72 ]. An empirical Bayes moderated t-test [ 72 , 73 ] was applied to detect differently expressed genes across treated and control samples. The generated gene list was further filtered for genes with low intensity and with small changes in expression. The IPA software is an online exploratory tool with a curated database for over 20, mammalian genes and 1. The output results are given scores and p-values that are computed based on the number of uploaded genes in the cluster or network and the size of the network or cluster in the Ingenuity knowledge database.

Scores for IPA networks are the negative logarithm of the p-value, indicating the likelihood of the focus genes genes uploaded to IPA in a network being found together due to random chance. To characterize in detail the process of cartilage and bone formation in zebrafish, we first wanted to examine the effects of chemical treatments known to affect skeletal development.

Treatment of zebrafish larvae with vitamin D VitD3 was previously shown to result in enhanced bone formation, while continuous treatment with parathyroid hormone PTH led to decreased bone formation [ 63 ]. We decided to confirm and extend these findings by comparing the effects on skeletal formation to those on gene expression. VitD3 and PTH treatments were performed continuously from 5dpf to 10dpf. Control and treated larvae were stained by Alcian blue for cartilage extracellular matrix ECM and with Alizarin red to detect the calcified bone matrix.

At this stage, the head cartilage is well formed and a complete set of cartilage elements is observed Fig 1A and 1B. In contrast, although ossification begins at 3dpf and the first bone structures are visible at 5dpf, the bone skeleton continues its formation until 30dpf [ 68 ]. Nevertheless, at 10dpf, a number of bone elements are observed in the head region, the first vertebral centrae are formed, while others only begin to be calcified for example the branchiostegal ray2 Fig 1C and 1D.

In three independent experiments, 27—29 ventral view images of Alcian blue- or Alizarin red-stained larvae were obtained. After 5days of VitD3 or PTH treatment, cartilage stays unchanged as compared to the control by general observation. The structures are well formed, complete with the glycosaminoglycans present in the cartilage matrix judging from the similar staining intensity Fig 1E—1G.

In a close-up view Fig 1E—1G , inlays , no difference could be observed in cell shape or size between the different treatments. Considering bone calcification, a general observation revealed a clear increase of bone development upon VitD3 treatment Fig 1I. Some structures appear in advance, such as the retroarticular Fig 1I arrowhead bone and the preopercular not shown bone, while some other structures are thicker such as the dentary or the ceratohyal, or longer such as the branchiostegal ray2.

Nevertheless, the general morphology was unchanged. In contrast, continuous PTH treatment led to a general decrease of bone formation and to a complete absence of some structures, such as the anguloarticulars and branchiostegal ray2 Fig 1J. Based on these images, we applied two complementary approaches to obtain a more objective qualitative and quantitative description of the skeleton.

The first one is a morphometric approach that evaluates the general aspect of the head skeleton by measuring the distances between and the relative position of all detected bone elements. The images were introduced into the CYTOMINE software see Materials and Methods , [ 69 ] and each image was annotated by positioning specific landmarks representing the different skeletal elements. For larvae stained for cartilage, 21 landmarks were defined Fig 1A , while 29 points of interest were positioned within the Alizarin red-stained bone skeleton Fig 1C.

In these pictures, we consider the head separated horizontally in 2 parts. Some structures are unique and located on the symmetry axis, while others are paired and localized symmetrically, such as the dentary, maxilla, entopterygoid, and hyosymplectic. The software then computes the distances between selected landmarks and the angles formed by lines drawn between selected points.

Fish Physiology Zebrafish, Volume 29

Morphometric analysis in VitD3-treated larvae cartilage revealed an increase of the distance between articulation ar "up" and "down", leading to a broader jaw as compared to untreated animals, while all the other distances remained unchanged S1A and S1C Fig. Morphometric cartilage analysis of larvae treated with PTH for 5 days revealed an increase in length of the ceratohyal cartilages ch, S1B Fig.

Analysis of the bone skeleton after VitD3 treatment revealed a significant increase of the distance between maxillae m, Fig 2A , consistent with a broader jaw as already observed by cartilage morphometry. The length of the head skeleton is also increased upon VitD3 treatment with a longer distance between the anterior part of the head an and the notochord n or the parasphenoid p. Other measures are not significantly modified Fig 2A and 2C. PTH treatment led to a significant decrease of the size of the parasphenoid p, Fig 2C.

The distances are measured in pixels. A Distances after VitD3 treatment. B Distances after PTH treatment. Abbreviations as in Fig 1. A Analysis of the bone skeleton after VitD3 treatment revealed a significant increase of the distance between maxillae m , consistent with a broader jaw as already observed by cartilage morphometry. The length of the head skeleton is also increased upon VitD3 treatment with a longer distance between the anterior part of the head an and the notochord n , and between an and the parasphenoid p bone. Other measures are not significantly modified A, C.

However, a significant broadening of the posterior head skeleton is revealed by the increased distance between left and right "up" and "down" branchiostegal rays1 br1 , entopterygoids en and also the opercula o B. The second approach consists in the evaluation of the intensity and progression of bone formation of the different bone structures, and their level of ossification. In each image, every bone structure is assigned a score, ranging from absent red , early ossification yellow , advanced green or over-ossified purple in comparison to a typical image of a control larva of the same age.

The distribution of the scores obtained for the different elements in VitD3- or PTH-treated larvae and the corresponding controls is shown in Fig 3 and the results of the statistical analysis are given in S2 and S3 Tables. 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 to the control; purple.

As no significant difference was observed for paired structures between left and right up and down , their scores have been combined. A Cumulated frequency after 5days VitD3 treatment. To obtain this, values were attributed to each element according to its category and added up for each larva: 0 for absent, 1 for early, 2 for advanced, and 4 for over ossification B Cumulated frequency after 5days PTH treatment. After 5 days VitD3 treatment, all the structures are present and some are over-ossified like the hyomandibular, the entopterygoid, the dentary and the ceratohyal bones.

Early delayed ossification is decreased for all the structures shown, as compared to controls, while advanced ossification increased in the maxilla, branchiostegal ray1, branchiostegal ray2 and anguloarticular Fig 3A. Statistical analysis S2 Table reveals that only the anguloarticular and the maxilla up do not change significantly in this condition. All the other structures br1, br2, m down, ch, d, en, hm are significantly increased, with the hyomandibulars, entopterygoids and ceratohyals displaying the most drastic effect.


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These results confirm a very significant positive effect of VitD3 treatment on bone formation. PTH treatment resulted in nearly opposite effects to VitD3. In summary, these complete image analyses reveals that VitD3 treatment conserves the general skeletal morphology, but leads to a longer head and a larger jaw. Bone calcification is stronger for most elements, and some elements calcify earlier. In contrast, PTH treatment conserves the general cartilage morphology except for an increased length of the ceratohyal.

In bone, PTH treatment leads to a general decrease of ossification. Some structures are missing and the parasphenoid is significantly decreased. To gain deeper insight into the molecular mechanisms involved in the observed skeletal modifications, we analyzed the expression of several genes selected for their known function in bone formation. One class of genes codes for structural proteins such as collagens Col1a1, Col1a2, Col10a1a or bone specific ECM proteins such as secreted acidic cysteine rich protein Sparc, previously named osteonectin or Osn , secreted phosphoprotein 1 Spp1, previously named osteopontin or Osp and bone gamma-carboxyglutamate protein Bglap, previously named osteocalcin or Ocn.

The second class of interest consists of those genes coding for factors involved in regulation of cartilage and bone differentiation, including the pth1a gene coding for PTH as well as transcription factor genes dlx5a , dlx6a , runx2b and osx. We first decided to follow the expression of these genes during the 6—10dpf period in untreated animals, using the glyceraldehydephosphate dehydrogenase gapdh house-keeping gene as reference selected from 3 candidate housekeeping genes, see Materials and Methods. Compared to their expression at 6dpf, we observe an increase of sparc , bglap , spp1 and col1a1 at 7dpf, followed by a decrease at 8dpf for sparc , bglap and spp1 , while the col1a1 gene peaked at 8dpf and decreased its expression at later stages Fig 4A and 4D.

The pth1a gene expression strongly increased fold during the 6—10dpf period, while runx2b displayed a fold increase. The transcription factor gene dlx5a displayed an expression peak at 7 and 8dpf and decreased after that, while dlx6a was unaffected and osx surprisingly revealed a 2-fold decrease from 6 to 7dpf Fig 4G and 4J. A,D,G,J Specific mRNA levels at 6dpf relative to the gapdh house-keeping gene were used as reference, and then compared to the corresponding level in larvae of different age.

E-F,H-I,K-L Specific mRNA levels in treated larvae were determined relative to the gapdh reference house-keeping gene and then compared to the corresponding level in untreated controls of the same age. A-C Bone matrix markers bglap , sparc , spp1. D-F Collagens col1a1 , col1a2 , col10a1a. G-I Transcription factors dlx5a , dlx6a and osx. J-L Parathyroid hormone pth1a and transcription factor runx2b. We then investigated the modulation of expression of these genes during drug treatment starting at 5dpf. Compared to untreated controls, VitD3 treatment led to a clear and significant increase in expression of all the structural protein genes: sparc , bglap , spp1 , col1a1 and, to a lesser extent col1a2 and col10a1a Fig 4C and 4F.

These results correlate well with the observed increase in bone calcification observed at 10dpf. Among the regulatory factor genes, only pth1a revealed a strong up-regulation that increased during the treatment, while dlx5a and dlx6a were transiently induced at 8 and 9dpf. Finally, runx2b displayed a weak but significant increase up to 1.

On the other hand, relative to untreated controls, PTH treatment resulted in a transient increase of spp1 at 8—9dpf, while sparc , and bglap were unchanged before a decrease at 10dpf Fig 4B. Surprisingly, no significant effect of PTH treatment was observed on the expression of the collagen genes Fig 4E. Among the regulatory factors, osx expression remained constant, while pth1a , dlx5a , dlx6a and runx2b declined at 10 dpf Fig 4H and 4K. Taken together, these observations are consistent with the observed decrease in bone matrix calcification at 10dpf. To obtain a global view of the physiological changes caused by PTH and VitD3 treatment, we performed a microarray whole genome expression analysis.

We compared 6dpf control larvae to larvae treated between 5dpf and 6dpf with the corresponding compounds, in order to capture early regulatory events rather then secondary regulations leading ultimately to the observed modulations of bone formation at 10dpf.

Four independent experiments were carried out and total RNA was extracted from control and VitD3-treated 6dpf larvae. A complete list of genes affected more than 1. Six genes were selected from the list for validation by RT-qPCR, which demonstrated the reliability of the microarray data Table 1. Confirming that the VitD3 pathway was indeed activated, the most highly induced gene is cyp24a1 , encoding a member of the cytochrome P superfamily of enzymes involved in the degradation of 1,dihydroxyvitamine D3.

Modulation of the insulin pathway is indicated by the significant induction of igfbp1 and igf2. According to Ingenuity Pathway Analysis IPA; Materials and Methods , other biological functions that were affected by vitamin D treatment S5 Table are related to lipid, small molecule, amino acid, carbohydrate and drug metabolism, followed by organismal and cardiovascular system development. A striking feature of the affected genes list is the abundance of genes involved in molecular transport, from ion channels to ATP-dependent pumps S4 Table , consistent with a profound adaptation to the changes in metabolism that were also previously observed [ 75 — 77 ].

Among the transcription regulatory factors, we note the decreased expression of ppara and of foxo3 , involved in lipid metabolism, as well as fosb and twist1 , while klf11 and klf13 were significantly induced S4 Table. A network of regulatory interactions could be constructed, comprising genes common to both systems and genes specific for each organ S2 Fig.

Interestingly, muscle structural genes such as TTN Titine are inhibited. PTH treatment between 5dpf and 6dpf resulted in less modulation of gene expression S6 Table. Six genes were selected from the list to include up- and down-regulated genes for independent confirmation of the microarray expression results by RT-qPCR Table 1.

Interestingly, we observed a decrease 2. Additional affected genes are the repressed cyp21a2 and hsd3b7 , indicating a decrease in steroid degradation. IPA comparison between PTH and VitD3 effects reveals that, unlike the general metabolic effects exerted by VitD3, the most prominent biological functions affected by PTH treatment were related to cell development, signaling and embryonic development S7 Table. The most highly developmentally affected systems were hematopoiesis and the skeletal, muscular and cardiovascular systems.

Comparison of the genes affected by the two hormones exerting opposite effects on bone formation, VitD3 and PTH, revealed only 12 genes in common Fig 5A and 5B. Using these 12 genes allows building a regulatory network around the protooncogene MYC and containing several genes that are differentially regulated in these two conditions Fig 5A and 5C , suggesting opposing effects on mitochondrial GSR , pyrimidine CAD or lipid metabolism CES1.

A List of common genes and their respective log2 fold change in the two conditions. The number of probes resulting in different hybridization signals is given, with the numbers in parenthesis and the graph showing the numbers of IPA-annotated genes. C Network constructed using the common genes and extended using the genes affected in one of the two conditions.

In a first experiment, zebrafish larvae were grown at normal gravity 1g until 5dpf. One half of the population was brought to 3g hypergravity in the LDC for another 4 days, while the other half was kept at 1g see Fig 6. At 9dpf, the larvae were stained with Alizarin red for bone matrix Fig 7A and 7B and analyzed as described above. No difference was observed between the two samples when total length of the larvae or size of the eye or lens was determined not shown. In the morphometric analysis, the 3g larvae present a larger head skeleton with a significant increase of the distance between the 2 anguloarticular bones, branchiostegal rays1, entopterygoid and the opercles Fig 7C.

In bone formation analysis S3A Fig , S8 Table , the anguloarticular, branchiostegal ray2 and hyomandibular presented a clear over ossification, while the ceratohyal presented a significantly higher proportion of advanced ossification. In contrast, the dentary, maxilla and entopterygoid were not significantly affected S3A Fig. A clearly weaker calcification was observed in the otoliths. A larvae are placed at hypergravity at 5dpf until 9dpf 3g , while control larvae are kept at normal gravity for 9 days.

Total mRNA was extracted at 6dpf and batches of larvae were fixed at 9dpf for Alizarin red staining of bone matrix. An additional batch of larvae was kept at normal gravity until 6dpf 1g. RNA extraction and Alizarin red staining are performed at 6dpf. For abbreviations see legend to Fig 1. A,B Alizarin red staining of 9dpf control larvae A and larvae treated for 4 days in 3g hypergravity after 5 days at 1g B. Ventral view, anterior to the left.

C Comparison of morphometric measurements for some selected distances within the heads of control and 3g-treated larvae. D Global score for bone formation in control and 3g treated larvae. E Comparison of cumulated frequencies of, respectively light, 1 pair dark or two pairs dark otoliths in control and 3g treated larvae. In addition, total mRNA was extracted from the larvae at 6dpf and whole genome gene expression was compared between larvae exposed for 1 day to 3g and 1g controls. The number of genes found to be modulated by hypergravity was , although the extent of induction or repression was surprisingly low S9 Table , but significant as confirmed by RT-qPCR for 5 selected genes S10 Table.

Interestingly, among the affected biological functions S11 Table , cellular and organism developmental processes ranked highest, only molecular transport appears in second position. More specifically, development and function of the skeletal and muscular system and connective tissue ranked highest, followed by the nervous and endocrine systems and finally hematological and cardiovascular systems. Among the specifically affected genes, many transporter and ion channel genes are present, reminiscent of the observations after VitD3 treatment.

Interestingly, among the transcription factors, vitamin D receptor vdr is weakly, but significantly down-regulated, similar to the nuclear receptor pparg. Other prominent transcription factor genes are the homeo-box containing pou3f3 and its potential partners meis1 and onecut1. As an approach to investigate some of the effects on zebrafish physiology to be expected when going into real microgravity, we applied a protocol that we would qualify as "Reduced Gravity Paradigm" or "relative microgravity". The principle is to grow the zebrafish larvae for a defined period 5 days in a hypergravity environment this case 3g , before returning them to normal gravity for one additional day Fig 6B.

The effect of this decrease in gravity on bone formation and gene expression was then investigated. Zebrafish fertilized eggs were subjected at 4hpf to 3g hypergravity until 5dpf. For comparison, a parallel batch was grown at normal gravity outside of the centrifuge chamber 1g. The morphology of the embryos and larvae was monitored every day by microscopic observation, no striking effect was observed on developmental processes such as segmentation, organogenesis or hatching time.

Only a clearly decreased delayed pigmentation was observed at 24hpf Fig 8A and 8B , which was rapidly resolved as pigmentation was indistinguishable in 1g and 3g embryos at 2dpf. A, B comparison of pigmentation at 24hpf in 1g A and 3g B larvae. G Global scores for bone formation in control and the different treated larvae. At 5dpf, the larvae exposed for 5 days to 3g in the LDC, were separated in three distinct batches, one was left in the LDC for another day 3g while the other two were returned to normal gravity for one day. The 1g batch continued to grow at normal gravity outside of the centrifuge chamber for the entire 6 days.

At 6dpf, all larvae were collected and stained for calcified structures using Alizarin red. Compared to larvae grown for 6 days at 1g, the bone structures in the head of all 3g exposed larvae appeared more intense Fig 8C—8F , more specifically the anguloarticular, maxillary and, to a lesser extent the ceratohyal, hyomandibular and branchiostegal ray 1 S12 Table. The global score was significantly increased in all samples exposed to 3g for 5 or 6 days Fig 8G.

Morphological analysis revealed a significant increase in the distance between branchiostegal rays 1, enteropterygoids and opercles, and an increase in the parasphenoid area Fig 9. A Distances between the different cranial bone elements. B Area of the parasphenoid bone. At 6dpf, all larvae were collected and used for mRNA extraction. Gene expression was determined by micro-array analysis, larvae exposed to 3g for the entire 6 days were chosen as control S13 — S15 Tables.

Relative to this hypergravity sample, a remarkable similarity was observed in the biological functions affected in the normal gravity larvae Table 2. Among the top ten functions modulated in each condition we found, on the one hand cell growth and proliferation, development, death and survival, organization and function, on the other hand embryonic and organismal organ development with a focus on connective tissue and cardiovascular development in the 6 days control at 1g.

Several genes, mostly common to all three conditions, were selected and the modulation of their expression was confirmed by RT-qPCR S16 Table. Most importantly, these central genes were affected to the same extent, relative to the 3g for 6 days control, whether the larvae were kept at 1g during the entire experiment or only for the last day, suggesting that their expression levels are specific to this gravitational condition and are rapidly within one day adapted to new conditions.

Further analyses were performed using all the genes common to any two of the conditions S5 Fig , also analyzed according to their potential function in individual organ systems S6 Fig. Expression of NFKBIA , a target gene for the NFkB pathway coding for an inhibitor of this pathway, was decreased in two conditions, potentially causing the decreased expression of the antiproliferative factor BTG2 [ 80 ] observed in all three conditions. The absolute number of probes resulting in a statistically significant hybridization signal is given for each condition.

In parentheses, the corresponding number of genes with an annotation in IPA is given, while the Venn diagrams represent the number of genes unique to each condition and genes common to two or three conditions. Color overlay indicates the fold change relative to the 3g sample taken as control. Among the affected genes, 41 were common to both experiments Fig 10 that could be assembled in a regulatory network S7 Fig. Zebrafish present remarkable degrees of similarity with mammals in the molecular mechanisms involved in their developmental biology and physiology.

Moreover, their ease of husbandry, high fecundity, and small size paves the way for a possible future space experiment, triggering the proposal of their use for the study of gravitational biology [ 83 — 89 ]. We decided to explore the effects of increased gravity hyper-g on zebrafish larvae using the large diameter centrifuge LDC. This device allows applying a well-controlled and constant centrifugal force 1gg by minimizing, through the large diameter of the rotating arms, the possible effects of Coriolis force [ 64 ]. Our aim was to concentrate on the effects on bone formation, therefore we chose to start the experiments at 5dpf, when perichondral ossification is taking place within all major cranial cartilage elements and intramembranous bone formation is ongoing.

We evaluated the effects on cartilage and bone formation by staining these structures after several days of treatment, at 9 or 10dpf. For a more detailed, more accurate and more objective evaluation of skeletal development, we developed two different, but complimentary methods for analyzing images of stained zebrafish larvae.

The first one uses a number of landmarks placed manually within the images using the software environment CYTOMINE and allows automatic extraction of distances and angles between these landmarks, ultimately resulting in a morphometric description of the head skeleton. The second one is based on manually assigning a developmental score to each cranial bone element within each image, enabling us to calculate a mean score for each element and a global score for each individual. To validate these approaches, we performed two treatments of zebrafish larvae whose effects had been previously described [ 63 ].

The first treatment uses exogenous vitamin D3 VitD3 [ 90 ] to increase bone formation, indeed the general VitD3 metabolism in teleosts is similar to that in mammals, teleosts possess two vitamin D receptors VDRs and knock-down of VDRa expression causes a decrease of calcium ion uptake [ 90 ]. PTH and related peptides are known hypercalcemic agents in mammals, however their function is more controversial in teleosts, depending on the species [ 91 ].

Although teleosts do not present a parathyroid gland, they do produce PTH in the gills, probably in cells identified by the expression of gcm2 , a gene whose orthologues are required for parathyroid development in chicken and mouse [ 92 , 93 ]. PTH administration induced hypercalcemia in fugu Tetraodon nigrividans by inducing both osteoblast and osteoclast function and by decreasing scale calcium content [ 94 ].

Genes homologous to the mammalian PTH-related peptides PTHrP were found in teleosts, they are more widely expressed [ 95 ], they increase calcium uptake in sturgeon Acipenser nacarii [ 96 ]and were shown to play different roles in craniofacial development in zebrafish [ 97 ]. Finally, four stanniocalcin stc genes are present in fugu and zebrafish, only stc1-a expression was sensitive to the calcium concentration in water [ 99 ]. Depending on the mode of administration intermittent or continuous PTH and PTHrP were shown, respectively to increase or decrease bone formation in zebrafish [ ] or seabream [ ].

Fish Physiology: Zebrafish: Volume 29

We confirm the effects described in zebrafish on general bone formation and in addition, the combined approach allowed us give a more detailed description of these effects. Although the general morphology was preserved in both cases, VitD3 treatment lead to a broader jaw both in cartilage and bone and a longer head in bone, while PTH treatment leads to an increased length of the ceratohyal cartilage, a general decrease of ossification, a decreased length of the parasphenoid bone and a broadening of the posterior head skeleton.

The discrepancy between cartilage and bone concerning the longer head probably results from the fact that the landmarks used in bone parasphenoid and notochord do not have a real equivalent in cartilage and may mineralize independently from it. Similarly, the developmental scoring method allowed a more differentiated description of the observed effects Fig While VitD3 treatment caused a generally significant increase in ossification of most elements, this was less prominent for the maxillary and absent for the anguloarticular.

Conversely, the decrease of ossification caused by PTH treatment was significant for all elements except branchiostegal rays 1. Understanding of the molecular mechanisms underlying these differential effects on the various skeletal elements and their morphology will require further investigation.

In red, the scores are significantly increased. In green, the scores are significantly decreased. A PTH. B VitD3. C 3g hypergravity between 5—6dpf D "relative microgravity". The decrease in otolith calcification was already previously described [ , ] and was proposed to involve a regulatory mechanism linking gravity sensing to the production of carbonic anhydrase and other matrix proteins in the inner ear [ — ].

Thus, the decrease in otolith calcification after prolonged exposure to 3g was expected, but it also emphasizes the specificity of the observed increase in ossification. During early exposure to 3g in the "relative microgravity" experiments , we observed a transient delay in pigmentation at 24hpf, which was rapidly resorbed at 48hpf. This finding is reminiscent of the transient decrease in the number of melanocytes that was observed at 24hpf during early exposure to simulated microgravity using a Rotating Wall Vessel device [ 51 ].

It is at present unclear whether a common mechanism may explain such a similar delay both in hypergravity and in simulated microgravity. We then turned to studying differences in gene expression caused by the various treatments. We chose to perform these studies using mRNA from entire larvae, as methods for isolation of specific cells, such as dissection or fluorescent cell sorting might not be available in a future space experiment.

First, we followed expression of bone-specific genes during normal development between 6 and 10dpf. We observed a sharp rise of mRNA coding for bone matrix proteins Sparc, Bglap, Spp1 and Col1a2 followed by a rapid decrease after 7dpf, suggesting that the major part of the bone matrix is formed at 7dpf and that further ossification is mainly due to mineral deposition. This is consistent with the observed sharp decrease of osx expression, followed with some delay by dlx5a expression, both indicating a decrease in osteoblast differentiation.

The continuous decrease in the levels of col10a1a mRNA could be related to the proposed inhibitory effect of this factor on biomineralization [ , ], while the large increase of runx2b and pth1a mRNA during the entire period could be related to some other functions of these factors [ , ]. Following the modulation of gene expression during chemical treatments revealed a clear upward trend for bone matrix protein-encoding genes upon VitD3 treatment and a clear downward trend during PTH treatment. These trends are consistent with the assumption that bone matrix secretion plays a functional role in the observed increase or decrease, respectively, in bone formation.

Expression of osx is increased during the first day of VitD3 treatment and decreased during PTH treatment, again consistent with a respectively prolonged or shortened period of osteoblast differentiation, also further supported by the increase of dlx5a and dlx6a expression at 8—9dpf during VitD3 treatment.

Finally, to determine the effects on whole genome gene expression of the various treatments, we chose to concentrate on mRNA levels only after one day of treatment, as we are mainly interested in regulatory events. A summary of all the genes affected by any of the studied conditions is shown in S17 Table. Again, we validated our approach by investigating the effects of known regulators of bone formation.

As expected, VitD3 treatment induced cyp24a1 expression, while PTH administration led to a decrease in endogenous pth1a expression. Furthermore, VitD3 treatment caused significant changes in overall metabolism, as shown by the involvement of affected genes in molecular transport or lipid metabolism. Probably for this reason, functions related to embryogenesis or organ morphology rank much lower in the list of affected pathways.

These findings are consistent with previous results, obtained using a deep sequencing RNA-seq approach, which also showed a high proportion of metabolic pathways affected by VitD3 treatment, administered either between 2 and 6—7dpf or between 6—7dpf [ ]. In contrast, PTH treatment affected less genes, but these were more involved in developmental processes. Interestingly, several genes were regulated in opposite directions upon VitD3 or PTH treatment Fig 5A and 5C , suggesting that they may be involved in the opposite effects on bone mineralization that we observed. However, when we classified the genes according to their known involvement in specific organ function S4 Fig , these genes were more specifically known for their function in muscle, indicating that further investigations are required.

When comparing genes and pathways affected by hypergravity, cellular growth and proliferation functions ranked very high, followed by cellular, tissue and organismal development Table 2. Among the canonical pathways affected S18 Table , we found those involving IGF, as already mentioned, and those involving pituitary hormones Prl and Gh as well as nuclear receptors.

Interestingly, finer analysis of the affected biological functions revealed that all hypergravity conditions acted on organism survival and cell apoptosis S19 Table , although no effect on larval survival or growth was observed in our experiments. Affected regulatory networks comprise PPARG, involved in adipocyte differentiation and regulating blood glucose uptake, consistent with the presence of other genes connected to insulin function. This observation may be related to previous experiments in rodents that showed a decrease in fat mass in hypergravity [ , ].

Fish Physiology: Zebrafish: Volume 29 : Steve F. Perry :

Another gene consistently induced by hypergravity in mammals is the Hsp70 stress response gene [ , ]. In zebrafish kept for the first two days at 3g, increased expression of a fluorescent reporter transgene hspgfp hypergravity was shown mainly in the lens [ 49 ], however no induction of the hsp70 gene was observed here, probably due to the later observation stages.

This indicates that older fish larvae are probably less stressed by hypergravity than are mammalian systems. Note that changes in the fli1-gfp transgene expression were also only observed for exposures before 24hpf [ ]. The c-FOS gene was first described as the cellular homolog of the viral oncogene causing murine osteosarcoma [ ], while gene knock-out mice suffered from severe defects in bone development and haematopoiesis [ ].

First microgravity experiments in murine carcinoma cells revealed a decreased induction of c-Fos and its heterodimeric partner c-Jun by growth factors [ , ]. Decreased c-Fos expression in microgravity was also observed in osteoblastic cells [ , ], while exposure to intense hypergravity 50—90g caused an increased expression of c-Fos and Egr1 [ ].

More moderate hypergravity conditions 3g also revealed rapid 36 min induction of c-Fos expression in osteoblasts [ ], while both hypergravity loading and unloading caused increased expression in rat brains [ , ]. This latter c-Fos induction was then considered as an indicator for neural activity in specific brain regions, in particular those related to vestibular sensing and processing [ — ].

Here, we show that exposure of zebrafish embryos to 3g hypergravity during the first 5—6 days of development leads to increased expression of fos , as part of a regulatory network composed of 6 other genes fosb , egr1 , edn1 , socs3a , gadd45b , klf2a that are induced in 3g conditions. Among these, the fos homolog fosb and the Zn-finger transcription factor gene egr1 belong to the immediate-early class of genes that are rapidly induced by growth factors. In mouse, FosB knock-out leads to behavioral defects [ ], while Egr1 null mice display sterility, impaired growth and pituitary development [ , ].

Egr1 was also rapidly induced in osteoblast cells upon mechanical stress [ ]. In zebrafish [ ], egr1 was shown to be part of a regulatory cascade controlling cartilage development [ ] that is induced by Fgf signaling [ ]. Edn1 is a vasoconstrictor peptide whose absence causes elevated blood pressure and craniofacial abnormalities [ ] in mouse, while a zebrafish edn1 mutant displayed mainly defects in cranial cartilage development [ 7 , ]. Socs3 is a suppressor of cytokine signaling; in mouse it was shown to inhibit placental and fetal liver erythropoiesis [ ], while a zebrafish mutant in the paralog socs3a was deficient in hair cell development and regeneration in the inner ear and the lateral line neuromasts [ ].

Gadd45b is a factor causing growth arrest upon DNA-damage, but also involved in hematopoiesis and immune response [ ]. Finally, loss of the Klf2 gene in mouse causes defects in vascular, skeletal and craniofacial development and in erythropoiesis [ ], while a zebrafish klf2a mutant displayed impaired cardiac valve development due to a deficient response to blood flow [ ]. Klf2a was further shown to be required for nitric oxyde NO synthesis during artery and hematopoietic stem cell development [ ], a process that is also highly involved in bone development [ — ].

Taken together, the network formed by these seven genes that are up-regulated in 3g conditions carries the potential to affect most processes that are known to be influenced by gravitational changes; from vestibular gravity sensing to hematopoiesis, immune response, vascular system and finally the skeletal system as was illustrated here. Moreover, this network is activated not only in larvae grown at 3g relative to larvae grown at 1g for 6 days, but also relative to larvae grown at 3g for 5 days and then returned to 1g for only one day Figs 10 and Increased expression of this gene network appears to be specific for hypergravity, while expression rapidly returns to normal after 1 day at 1g.

In mouse, Sox10 knock-out leads to neurological defects [ ], while sox10 mutant zebrafish are deficient in melanocyte pigmentation and inner ear development [ — ]. Similarly, Hes5 was shown to regulate neurogenesis [ ], but also human cartilage differentiation under the control of Notch signaling [ ]. Lgals3bp was shown to play a role in immune response and cell adhesion [ ]. HBE1 codes for one of the embryonic hemoglobins, suggesting alterations in oxygen transport under different gravity conditions.

MVP is a component of the ribonucleoprotein "vault" structures involved in nucleo-cytoplasmic transport and signal transduction [ ]. Interestingly, loss of function studies for Mvp in zebrafish revealed defects in brain development and the response to mechanical stimulus touch [ ]. The precise role of these genes in detection of decreased gravity and signal transmission to other physiological systems remains to be established.

Actually, the overall effect of 1 day exposure to 3g was surprisingly small at the genome level, compared to the other hypergravity experiments Tables S11 , S13 — S15 , a result that is reminiscent of that observed previously in mammalian renal cells [ ]. This observation suggests that the "Reduced Gravity Paradigm" is not simply a reversed hypergravity experiment, but rather that it represents a specific experimental condition.

Future experiments will reveal whether this approach may be considered as a good approximation of microgravity. In conclusion, we present an approach to objectively characterize cranial skeletal development in zebrafish larvae by morphometric image analysis and used this method to further characterize the effects of VitD3 and PTH on cartilage and bone formation.

Fish Physiology: Zebrafish, Volume 29

We have followed the expression of selected bone-related genes during 5 days of VitD3 or PTH treatment and analyzed whole genome gene expression after 1-day treatment. We have compared and correlated these results to the effects of hypergravity exposure on cranial skeleton formation. Finally, we have implemented a new type of hypergravity experiment, the "Reduced Gravity Paradigm", which allowed identification of a regulatory network of seven genes that are up-regulated in 3g, as well as several genes whose expression is rapidly modified when switching between 1g and 3g regimes.

Future investigations will reveal whether these gene regulations are specific for particular organ systems and how they contribute to the overall physiological adaptation to altered gravitational environments. A, C Distance after VitD3 treatment. Abbreviations as in 1.

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A Morphometric analysis in VitD3-treated larvae cartilage revealed an increase of the distance between articulation ar "up" and "down", leading to a broader jaw as compared to untreated animals, while A, C all the other distances remained unchanged. B Morphometric cartilage analysis of larvae treated with PTH for 5 days revealed a significant increase in length of the ceratohyal cartilages only D.

Genes filtered according to the described function for their human homologs using IPA in muscle or bone function. A Cumulated frequency after 3g between 5—9dpf.

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For abbreviations see legend to 1. Genes filtered according to the described function for their human homologs using IPA in bone, muscle, or cardiovascular system function. A network was constructed using the genes common to any two of the three experiments. Networks were constructed using the genes common to any two of the three experiments and filtered according to the described function for their human homologs using IPA in bone, muscle or cardiovascular system function.

A The bone structures distributed in 2 categories early and advanced ossification B The bone structures distributed in 3 categories early, advanced and over ossification. A The bone structures distributed in 2 categories early and advanced ossification B The bone structures distributed in 3 categories absent, early and advanced ossification. The indicates the human homolog of the gene, its "Entrez" gene name, the log ratio of VitD3-treated larvae compared to control, the presence of duplicate probes on the microarray D and the type of protein it encodes. Genes are arranged according to their type and in alphabetical order.

Ingenuity Pathway Analysis of the list of genes affected at 6dpf after VitD3 treatment for 24 hours. Columns indicate respectively the function, the range of p-values significance associated to various sub-functions, and the number of genes concerned N. The table indicates the human homolog of the gene, its "Entrez" gene name, the log ratio of PTH-treated larvae compared to control, the presence of duplicate probes on the microarray D and the type of protein it encodes.

The indicates the human homolog of the gene, its "Entrez" gene name, the log ratio compared to larvae kept at 1g between 0 and 6dpf, the presence of duplicate probes on the microarray D and the type of protein it encodes. The fold change and statistical significance p-values are given from the microarray data and the RT-qPCR confirmation experiments. Columns indicate respectively the category of function, the range of p-values significance associated to various sub-functions, and the number of genes concerned.

The indicates the human homolog of the gene, its "Entrez" gene name, the log ratio of 1g larvae compared to larvae kept at 3g between 0 and 6dpf, the presence of duplicate probes on the microarray D and the type of protein it encodes. The gene symbol and name is given, as well as the log fold-change values in the different experiments.

This work was supported by the "Fonds de la Recherche Fondamentale Collective"; 2. Alan Dowson for his support. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Teleost fish such as zebrafish Danio rerio are increasingly used for physiological, genetic and developmental studies. Help Centre. My Wishlist Sign In Join. Be the first to write a review. Add to Wishlist. This title is not in stock at the Booktopia Warehouse and needs to be ordered from our supplier.

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