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

Expression of osterix Is Regulated by FGF and Wnt/β-Catenin Signalling during Osteoblast Differentiation

Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF) and canonical Wingless-type MMTV integration site (Wnt/β-Catenin) signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx). We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/β-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation

Evaluation of a Water Channel-Based Platform for Characterizing Aerostat Flight Dynamics: A Case Study on a Lighter-Than-Air Wind Energy System

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140442/1/6.2014-2711.pd

Divergent transcriptional activities determine limb identity

Limbs develop using a common genetic programme despite widely differing morphologies. This programme is modulated by limb-restricted regulators such as hindlimb (HL) transcription factors Pitx1 and Tbx4 and the forelimb (FL) Tbx5. Both Tbx factors have been implicated in limb patterning and growth, but their relative activities and underlying mechanisms remain unclear. In this paper, we show that Tbx4 and Tbx5 harbour conserved and divergent transcriptional regulatory domains that account for their roles in limb development. In particular, both factors share an activator domain and the ability to stimulate limb growth. However, we find that Tbx4 is the primary effector of HL identity for both skeletal and muscle development; this activity relies on a repressor domain that is inactivated by a human TBX4 small-patella syndrome mutation. We propose that limb identity is largely achieved by default in FL, whereas a specific repressor activity unique to Tbx4 determines HL identity

Dcc Regulates Asymmetric Outgrowth of Forebrain Neurons in Zebrafish

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates

Mutations in fam20b and xylt1 Reveal That Cartilage Matrix Controls Timing of Endochondral Ossification by Inhibiting Chondrocyte Maturation

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed “maturation,” when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development

Mouse anatomy ontologies:enhancements and tools for exploring and integrating biomedical data

Mouse anatomy ontologies provide standard nomenclature for describing normal and mutant mouse anatomy, and are essential for the description and integration of data directly related to anatomy such as gene expression patterns. Building on our previous work on anatomical ontologies for the embryonic and adult mouse, we have recently developed a new and substantially revised anatomical ontology covering all life stages of the mouse. Anatomical terms are organized in complex hierarchies enabling multiple relationships between terms. Tissue classification as well as partonomic, developmental, and other types of relationships can be represented. Hierarchies for specific developmental stages can also be derived. The ontology forms the core of the eMouse Atlas Project (EMAP) and is used extensively for annotating and integrating gene expression patterns and other data by the Gene Expression Database (GXD), the eMouse Atlas of Gene Expression (EMAGE) and other database resources. Here we illustrate the evolution of the developmental and adult mouse anatomical ontologies toward one combined system. We report on recent ontology enhancements, describe the current status, and discuss future plans for mouse anatomy ontology development and application in integrating data resources. Mamm Genome 2015 Oct; 26(9-10):422-3

Marcadores do metabolismo ósseo em gatos

A formação e reabsorção óssea são processos contínuos no organismo e uma das maneiras de avaliar as alterações, a intensidade ou alterações desse processo é pela mensuração dos marcadores do metabolismo ósseo no soro ou na urina. Os objetivos deste estudo foram identificar se os testes comerciais para detectar osteocalcina (OC), peptídeo aminoterminal do procolágeno tipo I (PINP) e telopeptídeo carboxiterminal do colágeno tipo I (ICTP) em seres humanos podem ser utilizados em gatos; e mensurar os valores de OC, PINP e ICTP e correlacioná-los com a densitometria mineral óssea (DMO) em gatos adultos jovens. As médias das concentrações séricas da OC e do ICTP obtidas foram, respectivamente, 0,15±0,03ng mL-1 e 8,59±5,78ng mL-1. A média (±DP) da DMO apresentada foi 1,40±0,23mmAl. Os marcadores do metabolismo ósseo apresentaram baixa correlação entre si e com a DMO. Este estudo concluiu que o teste comercial para mensuração da OC e ICTP em seres humanos podem ser utilizados em gatos, e o teste comercial para mensuração do PINP em seres humano não é viável para essa espécie.Bone formation and resorption are continuous processes and a tool to evaluate alterations these activities is to measure seric or urinary markers of bone metabolism. The aims of this study were to identify if commercial sets available to detect human osteocalcin, (OC) amino-terminal propeptides of procollagen type I (PINP), type-I collagen carboxi-terminal telopeptide (ICTP) are also available for usage in domestic cats; measure seric levels of OC, PINP and ICTP; correlate findings with bone mineral density (BMD) in young adult cats. OC and ICTP means (±SD) was 0.15±0.03ng mL-1 and 8.59±5.78ng mL-1, respectively. BMD while 1.40±0.23mm Al. Bone metabolism markers showed low correlation between then and also BMD. This paper concludes that commercial sets designed for OC and ICTP measures in humans may be used for same purpose in cats to evaluate bone metabolism activity alterations and that kits used for PINP in human should not be used to evaluate in cats

Optimised isolation method for RNA extraction suitable for RNA sequencing from feline teeth collected in a clinical setting and at post mortem

Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of the microenvironment in many tissues. It is challenging to obtain high quality RNA from mineralised tissues. We developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Teeth were homogenised in phenol-guanidinium solution at near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced good RNA yields and improved RNA quality based on RNA integrity numbers equivalent (RINe) from an average of 3.6 to 5.6. No correlation was found between RNA purity parameters measured by A260:280 or A230:260 ratios and degree of RNA degradation. This implies that RNA purity indicators cannot be reliably used as parameters of RNA integrity. Two reference genes (GAPDH, RPS19) showed significant changes in expression levels by qPCR at low and moderate RINe values, while RPL17 was stable at all RINe values tested. Furthermore, we investigated the effect of quantity and quality of RNA on the quality of the resultant RNA sequencing (RNA-Seq) data. Thirteen RNA-seq data showed similar duplication and mapping rates (94 to 95%) against the feline genome regardless of RINe values. However one low yield sample with a high RINe value showed a high duplication rate and it was an outlier on the RNA-seq multidimensional scaling plot. We conclude that the overall yield of RNA was more important than quality of RNA for RNA-seq quality control. These results will guide researchers who wish to perform RNA extractions from mineralised tissues, especially if collecting in a clinical setting with the recognised restraints that this imposes