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