Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Background The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration

Putting crystals in place - the regulation of biomineralization in zebrafish

In humans the skeleton has a number of crucial functions: It provides protection and mechanical support to the body, is an important metabolic organ and represents the place of adult hematopoiesis. There are a number of human diseases related to the muscoskeletal system; prominent examples are osteoporosis and osteoarthritis. Osteoporosis is characterized by a gradual reduction of bone mass due to an imbalance between bone formation and resorption, which leads to an increased risk of fracture. In osteoarthritis, loss of joint cartilage tissue with subsequent inflammation and formation of bone spurs leads to chronic pain and loss of joint flexibility. These diseases represent a major burden to the health systems in an ever-aging society. Another aspect related to skeletal biology is the regulation of bio-mineralization. Ectopic (displaced) mineralizations can occur in most soft tissues and are a burden for patients with systemic mineral imbalance such as in chronic kidney disease, but are also seen as a consequence of rare, monogenetic diseases, injury or aging. Tissues of the cardiovascular system are particularly inclined to ectopic mineralization. Such ectopic mineralization in the cardio-vasculature correlates with severe clinical symptoms such as myocardial infarction. In recent years it has become clear that, mechanistically, bio-mineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. The research described in my thesis investigated zebrafish mutants showing defective bone-mineralization. The mutant “no bone” (nob), completely lacks mineralization, and the mutant “dragonfish” (dgf), shows excessive and pathologic mineralization in multiple tissues. The respective mutations could be attributed to the genes entpd5 (nob) and enpp1 (dgf), respectively. The well-established role of enpp1 as an enzyme generating pyrophosphate, allowed us to characterize entpd5, as a previously unknown and critical factor regulating bio-mineralization in zebrafish via the phosphate/pyrophosphate axis. A further key finding was that in response to ectopic mineralization in dgf mutants a rapid cellular response occurs by cells with osteoclastic (mineral resorbing) properties. This finding is of relevance for patients with a disease named generalized-arterial-calcification-of-infancy (GACI), which is caused by mutations in ENPP1. One treatment option for them is bisphosphonates which can inhibit further mineralizations, but are also known for their inhibitory effect on osteoclasts, which is exploited in the treatment of osteoporosis. Hence novel drugs inhibiting mineralization are desirable. We show in a proof-of-concept that dgf mutants can be a useful and easy to evaluate model for testing candidate compounds. Additionally we describe a zebrafish mutant for osterix, a key transcription factor in osteoblasts; and discovered that while the formation of the head skeleton in zebrafish depends on osterix, the first mineralized pattern in the axial skeleton occurs independently of osterix, pointing towards the existence of a distinct population of osteogenic cells that differs from the typical osteoblast

Not All Bones are Created Equal - Using Zebrafish and Other Teleost Species in Osteogenesis Research

Developmental osteogenesis and pathologies of mineralized tissues are areas of intense investigations in the mammalian field, but different from other areas of organ formation and developmental biology, zebrafish have been somewhat slow in joining the area of bone research. In recent years, however, genetic screens have provided a number of exciting mutants, and transgenic lines have been developed that permit visualization of osteoblasts and osteoclasts in vivo. We here review some of the recent literature and provide examples where insights from studies in zebrafish have complemented the information available from mammalian models or clinical studies. Furthermore, we provide a comparative overview about different forms of bone within the teleost lineage, and between teleosts and mammal

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression