Adelbert De Clercq

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.

Marfan syndrome (MFS) is a heritable connective tissue disorder caused by pathogenic variants in the gene coding for the extracellular matrix protein fibrillin-1. While the disease affects multiple organ systems, the most life-threatening manifestations are aortic aneurysms leading to dissection and rupture. Other cardiovascular complications, including mitral valve prolapse, primary cardiomyopathy, and arrhythmia, also occur more frequently in patients with MFS. The standard medical care relies on cardiovascular imaging at regular intervals, along with pharmacological treatment with β-adrenergic receptor blockers aimed at reducing the aortic growth rate. When aortic dilatation reaches a threshold associated with increased risk of dissection, prophylactic surgical aortic replacement is performed. Although current clinical management has significantly improved the life expectancy of patients with MFS, no cure is available and fatal complications still occur, underscoring the need for new treatment options. In recent years, preclinical studies have identified a number of potentially promising therapeutic targets. Nevertheless, the translation of these results into clinical practice has remained challenging. In this review, we present an overview of the currently available knowledge regarding the underlying pathophysiological processes associated with MFS cardiovascular pathology. We then summarize the treatment options that have been developed based on this knowledge and are currently in different stages of preclinical or clinical development, provide a critical review of the limitations of current studies and highlight potential opportunities for future research.

Genome‐wide association studies (GWAS) have improved our understanding of the genetic architecture of common, complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis‐related traits there is a need for efficient and cost‐effective in vivo functional testing. This can be achieved through CRISPR‐based reverse genetic screens, where phenotyping is traditionally performed in stable germline KO mutants. Recently it was shown that first‐generation (F0) mosaic mutant zebrafish (so‐called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss‐of‐function mutations in LRP5, a co‐receptor in the Wnt signaling pathway, cause Osteoporosis‐pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis‐related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro‐computed tomography (microCT) scanning and mass‐spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next‐generation sequencing analysis of adult crispant tissue revealed a mean out‐of‐frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder, but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared to lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5‐related primary osteoporosis, that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes.

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human β3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

Variation of vertebral centra numbers is common in vertebrates. Likewise, the number of associated elements such as ribs and neural and haemal arches can vary and affect all regions of the vertebral column. In mammals, only the number of cervical vertebrae is invariable. Variation of total vertebral centra numbers is well documented in teleost fish, often related to temperature. Less information is available about which part of the vertebral column and which associated elements are liable to variation. Here, variation in number of vertebral centra and associated elements is studied in Chinook salmon in six distinct anatomical regions. Animals are raised at 8 and 12°C to ask whether the vertebral centrum numbers, the pattern, and the frequency of variation in particular regions are temperature dependent. No significant difference concerning the total number of vertebrae was found, but regional differences occurred between the 8 and 12°C groups. Twelve specimens out of 60 of the 12°C group had three postcranial vertebrae compared to only one specimen in the 8°C group. The number of transitional vertebrae is significantly different in 8 and 12°C specimens. Fewer transitional vertebrae occur in more anterior positions in 8°C specimens. Most specimens of both temperature groups had two ural centra; however 17 specimens out of 60 of the 12°C group had up to five ural centra. Specimens of the 12°C group show more variation in the presence of the vestigial ribs associated with transitional vertebrae. Clearly, the postcranial, transitional, and ural regions are temperature sensitive. This study shows that nonsignificant differences in the total number of vertebrae can mask significant regional variation. Variation of vertebral numbers could be the consequence of loss or gain of vertebral centra and/or a change in the identity of the associated element on the vertebral centrum.

Skeletal deformities in farmed fish are a recurrent problem. External malformations are easily recognized, but there is little information on how external malformations relate to malformations of the axial skeleton: the external phenotype–skeleton link. Here, this link is studied in post‐hatch to first‐feed life stages of Chinook salmon (Oncorhynchus tshawytscha) raised at 4, 8 and 12°C. Specimens were whole‐mount‐stained for cartilage and bone, and analysed by histology. In all temperature groups, externally normal specimens can have internal malformations, predominantly fused vertebral centra. Conversely, externally malformed fish usually display internal malformations. Externally curled animals typically have malformed haemal and neural arches. External malformations affecting a single region (tail malformation and bent neck) relate to malformed notochords and early fusion of fused vertebral centra. The frequencies of internal malformations in both externally normal and malformed specimens show a U‐shaped response, with lowest frequency in 8°C specimens. The fused vertebral centra that occur in externally normal specimens represent a malformation that can be contained and could be carried through into harvest size animals. This study highlights the relationship between external phenotype and axial skeleton and may help to set the framework for the early identification of skeletal malformations on fish farms.

Teleost vertebral centra are often similar in size and shape, but vertebral‐associated elements, i.e. neural arches, haemal arches and ribs, show regional differences. Here we examine how the presence, absence and specific anatomical and histological characters of vertebral centra‐associated elements can be used to define vertebral column regions in juvenile Chinook salmon (Oncorhynchus tshawytscha). To investigate if the presence of regions within the vertebral column is independent of temperature, animals raised at 8 and 12 °C were studied at 1400 and 1530 degreedays, in the freshwater phase of the life cycle. Anatomy and composition of the skeletal tissues of the vertebral column were analysed using Alizarin red S whole‐mount staining and histological sections. Six regions, termed I–VI, are recognised in the vertebral column of specimens of both temperature groups. Postcranial vertebrae (region I) carry neural arches and parapophyses but lack ribs. Abdominal vertebrae (region II) carry neural arches and ribs that articulate with parapophyses. Elastic‐ and fibrohyaline cartilage and Sharpey's fibres connect the bone of the parapophyses to the bone of the ribs. In the transitional region (III) vertebrae carry neural arches and parapophyses change stepwise into haemal arches. Ribs decrease in size, anterior to posterior. Vestigial ribs remain attached to the haemal arches with Sharpey's fibres. Caudal vertebrae (region IV) carry neural and haemal arches and spines. Basidorsals and basiventrals are small and surrounded by cancellous bone. Preural vertebrae (region V) carry neural and haemal arches with modified neural and haemal spines to support the caudal fin. Ural vertebrae (region VI) carry hypurals and epurals that represent modified haemal and neural arches and spines, respectively. The postcranial and transitional vertebrae and their respective characters are usually recognised, but should be considered as regions within the vertebral column of teleosts because of their distinctive morphological characters. While the number of vertebrae within each region can vary, each of the six regions is recognised in specimens of both temperature groups. This refined identification of regionalisation in the vertebral column of Chinook salmon can help to address evolutionary developmental and functional questions, and to support applied research into this farmed species.

Polypterus senegalus is a freshwater fish belonging to the earliest diverged group of the actinopterygian lineage with currently living representatives. Its dentition has been well characterized in terms of distribution and shape of teeth in different life stages. Additionally, structural features of the first-generation teeth have been briefly described. However, at present it is not known how the primary dentition is patterned and if this pattern is similar to that found in juveniles. In this preliminary study, to answer this question we investigate the tooth pattern of a larval P. senegalus based on serial sections and 3D reconstructions, and compare dental features in larvae and juveniles. Polypterus senegalus passive aptero-larvae possess a well developed primary dentition. Dentigerous bones develop either prior to the teeth (dentary, retroarticular), or as an attachment site concomitant with the expanding dentition (coronoid 1). Most teeth are attached and erupted, and thus considered functional, and their pulp houses a blood vessel, two features that contrast with earlier findings. The primary teeth do not display a level of maturation that could reflect a particular order of development. New teeth are added anteriorly, and likely also posteriorly, to allow the tooth row to grow to keep pace with the growing jaw. Replacement teeth are absent at this stage. Thus, the pattern observed in juveniles, with alternate (odd and even) positions displaying a different level of tooth maturation, is not present in the larva. Most strikingly, in the larva, considerable symmetry exists between apparently random patterns on both dentaries, eliciting intriguing questions on left-right control to be addressed in future studies