Fish Model: Molecular and Cellular Basis of Bone Development and Homeostasis

A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 10543

Special Issue Editors

Laboratory for Organogenesis and Regeneration, Université de Liège, GIGA-R B34, Sart Tilman, 4000 Liège, Belgium
Interests: bone development; homeostasis; fish model
Research Group Evolutionary Developmental Biology, Ledeganckstraat 35, 9000 Gent, Belgium
Interests: evolutionary developmental biology; skeletal deformities; vertebral column

Special Issue Information

Dear Colleagues,

Skeletal pathologies are an increasing concern in our societies, including osteoporosis, osteopetrosis, craniofacial abnormalities, osteoarthritis, and vertebral deformities. They can be caused by genetic defects or predispositions but are also recognized to result from environmental or dietary influences. Fish are increasingly used as models with which to study skeletal development and homeostasis, including small fish species such as zebrafish and medaka for biomedical and genetic studies, as well as larger aquaculture species for assessing environmental factors. This Special Issue on fish models aims to emphasize the importance of these models for studying the molecular and cellular mechanisms underlying normal and pathological skeletal conditions. It will further present technical and methodological progress, as well as bottlenecks and limitations, in this field. In this context, we invite review articles that address the above-mentioned issues from a biological perspective and original research papers contributing to a better understanding of the processes involved.

Dr. Marc Muller
Prof. Dr. Eckhard Witten
Guest Editors

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Keywords

  • fish
  • bone
  • cartilage
  • malformations
  • homeostasis
  • genetics
  • environment

Published Papers (9 papers)

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Research

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26 pages, 4599 KiB  
Article
The Osteoblast Transcriptome in Developing Zebrafish Reveals Key Roles for Extracellular Matrix Proteins Col10a1a and Fbln1 in Skeletal Development and Homeostasis
Biomolecules 2024, 14(2), 139; https://doi.org/10.3390/biom14020139 - 23 Jan 2024
Viewed by 1299
Abstract
Zebrafish are now widely used to study skeletal development and bone-related diseases. To that end, understanding osteoblast differentiation and function, the expression of essential transcription factors, signaling molecules, and extracellular matrix proteins is crucial. We isolated Sp7-expressing osteoblasts from 4-day-old larvae using a [...] Read more.
Zebrafish are now widely used to study skeletal development and bone-related diseases. To that end, understanding osteoblast differentiation and function, the expression of essential transcription factors, signaling molecules, and extracellular matrix proteins is crucial. We isolated Sp7-expressing osteoblasts from 4-day-old larvae using a fluorescent reporter. We identified two distinct subpopulations and characterized their specific transcriptome as well as their structural, regulatory, and signaling profile. Based on their differential expression in these subpopulations, we generated mutants for the extracellular matrix protein genes col10a1a and fbln1 to study their functions. The col10a1a−/− mutant larvae display reduced chondrocranium size and decreased bone mineralization, while in adults a reduced vertebral thickness and tissue mineral density, and fusion of the caudal fin vertebrae were observed. In contrast, fbln1−/− mutants showed an increased mineralization of cranial elements and a reduced ceratohyal angle in larvae, while in adults a significantly increased vertebral centra thickness, length, volume, surface area, and tissue mineral density was observed. In addition, absence of the opercle specifically on the right side was observed. Transcriptomic analysis reveals up-regulation of genes involved in collagen biosynthesis and down-regulation of Fgf8 signaling in fbln1−/− mutants. Taken together, our results highlight the importance of bone extracellular matrix protein genes col10a1a and fbln1 in skeletal development and homeostasis. Full article
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34 pages, 3400 KiB  
Article
Exploring Omega-3′s Impact on the Expression of Bone-Related Genes in Meagre (Argyrosomus regius)
Biomolecules 2024, 14(1), 56; https://doi.org/10.3390/biom14010056 - 31 Dec 2023
Viewed by 866
Abstract
Dietary supplementation with Omega-3 fatty acids seems to promote skeletal health. Therefore, their consumption at imbalanced or excessive levels has offered less beneficial or even prejudicial effects. Fish produced in aquaculture regimes are prone to develop abnormal skeletons. Although larval cultures are usually [...] Read more.
Dietary supplementation with Omega-3 fatty acids seems to promote skeletal health. Therefore, their consumption at imbalanced or excessive levels has offered less beneficial or even prejudicial effects. Fish produced in aquaculture regimes are prone to develop abnormal skeletons. Although larval cultures are usually fed with diets supplemented with Omega-3 Long Chain Polyunsaturated fatty acids (LC-PUFAs), the lack of knowledge about the optimal requirements for fatty acids or about their impact on mechanisms that regulate skeletal development has impeded the design of diets that could improve bone formation during larval stages when the majority of skeletal anomalies appear. In this study, Argyrosomus regius larvae were fed different levels of Omega-3s (2.6% and 3.6% DW on diet) compared to a commercial diet. At 28 days after hatching (DAH), their transcriptomes were analyzed to study the modulation exerted in gene expression dynamics during larval development and identify impacted genes that can contribute to skeletal formation. Mainly, both levels of supplementation modulated bone-cell proliferation, the synthesis of bone components such as the extracellular matrix, and molecules involved in the interaction and signaling between bone components or in important cellular processes. The 2.6% level impacted several genes related to cartilage development, denoting a special impact on endochondral ossification, delaying this process. However, the 3.6% level seemed to accelerate this process by enhancing skeletal development. These results offered important insights into the impact of dietary Omega-3 LC-PUFAs on genes involved in the main molecular mechanism and cellular processes involved in skeletal development. Full article
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14 pages, 2529 KiB  
Article
Bone Formation in Zebrafish: The Significance of DAF-FM DA Staining for Nitric Oxide Detection
Biomolecules 2023, 13(12), 1780; https://doi.org/10.3390/biom13121780 - 12 Dec 2023
Viewed by 777
Abstract
DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the [...] Read more.
DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO. Full article
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18 pages, 4618 KiB  
Article
Loss of dlx5a/dlx6a Locus Alters Non-Canonical Wnt Signaling and Meckel’s Cartilage Morphology
Biomolecules 2023, 13(9), 1347; https://doi.org/10.3390/biom13091347 - 05 Sep 2023
Viewed by 767
Abstract
The dlx genes encode transcription factors that establish a proximal–distal polarity within neural crest cells to bestow a regional identity during craniofacial development. The expression regions of dlx paralogs are overlapping yet distinct within the zebrafish pharyngeal arches and may also be involved [...] Read more.
The dlx genes encode transcription factors that establish a proximal–distal polarity within neural crest cells to bestow a regional identity during craniofacial development. The expression regions of dlx paralogs are overlapping yet distinct within the zebrafish pharyngeal arches and may also be involved in progressive morphologic changes and organization of chondrocytes of the face. However, how each dlx paralog of dlx1a, dlx2a, dlx5a and dlx6a affects craniofacial development is still largely unknown. We report here that the average lengths of the Meckel’s, palatoquadrate and ceratohyal cartilages in different dlx mutants were altered. Mutants for dlx5a−/− and dlx5i6−/−, where the entire dlx5a/dlx6a locus was deleted, have the shortest lengths for all three structures at 5 days post fertilization (dpf). This phenotype was also observed in 14 dpf larvae. Loss of dlx5i6 also resulted in increased proliferation of neural crest cells and expression of chondrogenic markers. Additionally, altered expression and function of non-canonical Wnt signaling were observed in these mutants suggesting a novel interaction between dlx5i6 locus and non-canonical Wnt pathway regulating ventral cartilage morphogenesis. Full article
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18 pages, 6112 KiB  
Article
mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour
Biomolecules 2023, 13(5), 805; https://doi.org/10.3390/biom13050805 - 09 May 2023
Cited by 2 | Viewed by 1269
Abstract
The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were [...] Read more.
The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies. Full article
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30 pages, 4701 KiB  
Article
Elevated Water CO2 Can Prevent Dietary-Induced Osteomalacia in Post-Smolt Atlantic Salmon (Salmo salar, L.)
Biomolecules 2023, 13(4), 663; https://doi.org/10.3390/biom13040663 - 10 Apr 2023
Viewed by 1712
Abstract
Expansion of land-based systems in fish farms elevate the content of metabolic carbon dioxide (CO2) in the water. High CO2 is suggested to increase the bone mineral content in Atlantic salmon (Salmo salar, L.). Conversely, low dietary phosphorus [...] Read more.
Expansion of land-based systems in fish farms elevate the content of metabolic carbon dioxide (CO2) in the water. High CO2 is suggested to increase the bone mineral content in Atlantic salmon (Salmo salar, L.). Conversely, low dietary phosphorus (P) halts bone mineralization. This study examines if high CO2 can counteract reduced bone mineralization imposed by low dietary P intake. Atlantic salmon post-seawater transfer (initial weight 207.03 g) were fed diets containing 6.3 g/kg (0.5P), 9.0 g/kg (1P), or 26.8 g/kg (3P) total P for 13 weeks. Atlantic salmon from all dietary P groups were reared in seawater which was not injected with CO2 and contained a regular CO2 level (5 mg/L) or in seawater with injected CO2 thus raising the level to 20 mg/L. Atlantic salmon were analyzed for blood chemistry, bone mineral content, vertebral centra deformities, mechanical properties, bone matrix alterations, expression of bone mineralization, and P metabolism-related genes. High CO2 and high P reduced Atlantic salmon growth and feed intake. High CO2 increased bone mineralization when dietary P was low. Atlantic salmon fed with a low P diet downregulated the fgf23 expression in bone cells indicating an increased renal phosphate reabsorption. The current results suggest that reduced dietary P could be sufficient to maintain bone mineralization under conditions of elevated CO2. This opens up a possibility for lowering the dietary P content under certain farming conditions. Full article
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15 pages, 3798 KiB  
Article
Effect of Early Peptide Diets on Zebrafish Skeletal Development
Biomolecules 2023, 13(4), 659; https://doi.org/10.3390/biom13040659 - 07 Apr 2023
Cited by 3 | Viewed by 1360
Abstract
Incorporation of dietary peptides has been correlated with decreased presence of skeletal abnormalities in marine larvae. In an attempt to clarify the effect of smaller protein fractions on fish larval and post-larval skeleton, we designed three isoenergetic diets with partial substitution of their [...] Read more.
Incorporation of dietary peptides has been correlated with decreased presence of skeletal abnormalities in marine larvae. In an attempt to clarify the effect of smaller protein fractions on fish larval and post-larval skeleton, we designed three isoenergetic diets with partial substitution of their protein content with 0% (C), 6% (P6) and 12% (P12) shrimp di- and tripeptides. Experimental diets were tested in zebrafish under two regimes, with inclusion (ADF-Artemia and dry feed) or lack (DF-dry feed only) of live food. Results at the end of metamorphosis highlight the beneficial effect of P12 on growth, survival and early skeletal quality when dry diets are provided from first feeding (DF). Exclusive feeding with P12 also increased the musculoskeletal resistance of the post-larval skeleton against the swimming challenge test (SCT). On the contrary, Artemia inclusion (ADF) overruled any peptide effect in total fish performance. Given the unknown species’ larval nutrient requirements, a 12% dietary peptide incorporation is proposed for successful rearing without live food. A potential nutritional control of the larval and post-larval skeletal development even in aquaculture species is suggested. Limitations of the current molecular analysis are discussed to enable the future identification of the peptide-driven regulatory pathways. Full article
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Review

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18 pages, 3143 KiB  
Review
Recent Advances in Bioimage Analysis Methods for Detecting Skeletal Deformities in Biomedical and Aquaculture Fish Species
Biomolecules 2023, 13(12), 1797; https://doi.org/10.3390/biom13121797 - 14 Dec 2023
Viewed by 735
Abstract
Detecting skeletal or bone-related deformities in model and aquaculture fish is vital for numerous biomedical studies. In biomedical research, model fish with bone-related disorders are potential indicators of various chemically induced toxins in their environment or poor dietary conditions. In aquaculture, skeletal deformities [...] Read more.
Detecting skeletal or bone-related deformities in model and aquaculture fish is vital for numerous biomedical studies. In biomedical research, model fish with bone-related disorders are potential indicators of various chemically induced toxins in their environment or poor dietary conditions. In aquaculture, skeletal deformities are affecting fish health, and economic losses are incurred by fish farmers. This survey paper focuses on showcasing the cutting-edge image analysis tools and techniques based on artificial intelligence that are currently applied in the analysis of bone-related deformities in aquaculture and model fish. These methods and tools play a significant role in improving research by automating various aspects of the analysis. This paper also sheds light on some of the hurdles faced when dealing with high-content bioimages and explores potential solutions to overcome these challenges. Full article
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20 pages, 2251 KiB  
Review
Significance of Premature Vertebral Mineralization in Zebrafish Models in Mechanistic and Pharmaceutical Research on Hereditary Multisystem Diseases
Biomolecules 2023, 13(11), 1621; https://doi.org/10.3390/biom13111621 - 06 Nov 2023
Viewed by 1005
Abstract
Zebrafish are increasingly becoming an important model organism for studying the pathophysiological mechanisms of human diseases and investigating how these mechanisms can be effectively targeted using compounds that may open avenues to novel treatments for patients. The zebrafish skeleton has been particularly instrumental [...] Read more.
Zebrafish are increasingly becoming an important model organism for studying the pathophysiological mechanisms of human diseases and investigating how these mechanisms can be effectively targeted using compounds that may open avenues to novel treatments for patients. The zebrafish skeleton has been particularly instrumental in modeling bone diseases as—contrary to other model organisms—the lower load on the skeleton of an aquatic animal enables mutants to survive to early adulthood. In this respect, the axial skeletons of zebrafish have been a good read-out for congenital spinal deformities such as scoliosis and degenerative disorders such as osteoporosis and osteoarthritis, in which aberrant mineralization in humans is reflected in the respective zebrafish models. Interestingly, there have been several reports of hereditary multisystemic diseases that do not affect the vertebral column in human patients, while the corresponding zebrafish models systematically show anomalies in mineralization and morphology of the spine as their leading or, in some cases, only phenotype. In this review, we describe such examples, highlighting the underlying mechanisms, the already-used or potential power of these models to help us understand and amend the mineralization process, and the outstanding questions on how and why this specific axial type of aberrant mineralization occurs in these disease models. Full article
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