Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe

A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transfered and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involve

Visualization of Gli Activity in Craniofacial Tissues of Hedgehog-Pathway Reporter Transgenic Zebrafish

The Hedgehog (Hh)-signaling pathway plays a crucial role in the development and maintenance of multiple vertebrate and invertebrate organ systems. Gli transcription factors are regulated by Hh-signaling and act as downstream effectors of the pathway to activate Hh-target genes. Understanding the requirements for Hh-signaling in organisms can be gained by assessing Gli activity in a spatial and temporal fashion.We have generated a Gli-dependent (Gli-d) transgenic line, Tg(Gli-d:mCherry), that allows for rapid and simple detection of Hh-responding cell populations in both live and fixed zebrafish. This transgenic line expresses a mCherry reporter under the control of a Gli responsive promoter, which can be followed by using fluorescent microscopy and in situ hybridization. Expression of the mCherry transgene reporter during embryogenesis and early larval development faithfully replicated known expression domains of Hh-signaling in zebrafish, and abrogating Hh-signaling in transgenic fish resulted in the suppression of reporter expression. Moreover, ectopic shh expression in Tg(Glid:mCherry) fish led to increased transgene production. Using this transgenic line we investigated the nature of Hh-pathway response during early craniofacial development and determined that the neural crest skeletal precursors do not directly respond to Hh-signaling prior to 48 hours post fertilization, suggesting that earlier requirements for pathway activation in this population of facial skeleton precursors are indirect.We have determined that early Hh-signaling requirements in craniofacial development are indirect. We further demonstrate the Tg(Gli-d:mCherry) fish are a highly useful tool for studying Hh-signaling dependent processes during embryogenesis and larval stages

Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

<p>Abstract</p> <p>Background</p> <p>The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr></abbrgrp>. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study <it>chameleon </it>mutants, lacking a functional <it>disp1</it>(<it>con/disp1</it>).</p> <p>Results</p> <p><it>con/disp1 </it>mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in <it>con/disp1 </it>mutants, however <it>disp1 </it>is necessary for post-migratory CNCC patterning and differentiation. We show that <it>disp1 </it>is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, <it>sox9a </it>and <it>dlx2a</it>, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to inhibit Hh-signaling at different developmental stages, show that Hh-signaling is required during gastrulation for normal patterning of CNCC in the first PA, and then during the late pharyngula stage, to promote CNCC chondrogenesis within the posterior arches. Further, loss of <it>disp1 </it>disrupted normal expression of <it>bapx1 </it>and <it>gdf5</it>, markers of jaw joint patterning, thus resulting in jaw joint defects in <it>con/disp1 </it>mutant animals.</p> <p>Conclusion</p> <p>This study reveals novel requirements for Hh-signaling in the zebrafish PA skeleton and highlights the functional diversity and differential sensitivity of craniofacial tissues to Hh-signaling throughout the face, a finding that may help to explain the spectrum of human facial phenotypes characteristic of HPE.</p

Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

A New Mechanistic Scenario for the Origin and Evolution of Vertebrate Cartilage

The appearance of cellular cartilage was a defining event in vertebrate evolution because it made possible the physical expansion of the vertebrate “new head”. Despite its central role in vertebrate evolution, the origin of cellular cartilage has been difficult to understand. This is largely due to a lack of informative evolutionary intermediates linking vertebrate cellular cartilage to the acellular cartilage of invertebrate chordates. The basal jawless vertebrate, lamprey, has long been considered key to understanding the evolution of vertebrate cartilage. However, histological analyses of the lamprey head skeleton suggest it is composed of modern cellular cartilage and a putatively unrelated connective tissue called mucocartilage, with no obvious transitional tissue. Here we take a molecular approach to better understand the evolutionary relationships between lamprey cellular cartilage, gnathostome cellular cartilage, and lamprey mucocartilage. We find that despite overt histological similarity, lamprey and gnathostome cellular cartilage utilize divergent gene regulatory networks (GRNs). While the gnathostome cellular cartilage GRN broadly incorporates Runx, Barx, and Alx transcription factors, lamprey cellular cartilage does not express Runx or Barx, and only deploys Alx genes in certain regions. Furthermore, we find that lamprey mucocartilage, despite its distinctive mesenchymal morphology, deploys every component of the gnathostome cartilage GRN, albeit in different domains. Based on these findings, and previous work, we propose a stepwise model for the evolution of vertebrate cellular cartilage in which the appearance of a generic neural crest-derived skeletal tissue was followed by a phase of skeletal tissue diversification in early agnathans. In the gnathostome lineage, a single type of rigid cellular cartilage became dominant, replacing other skeletal tissues and evolving via gene cooption to become the definitive cellular cartilage of modern jawed vertebrates

Keratan sulphate in the tumour environment

Keratan sulphate (KS) is a bioactive glycosaminoglycan (GAG) of some complexity composed of the repeat disaccharide D-galactose β1→4 glycosidically linked to N-acetyl glucosamine. During the biosynthesis of KS, a family of glycosyltransferase and sulphotransferase enzymes act sequentially and in a coordinated fashion to add D-galactose (D-Gal) then N-acetyl glucosamine (GlcNAc) to a GlcNAc acceptor residue at the reducing terminus of a nascent KS chain to effect chain elongation. D-Gal and GlcNAc can both undergo sulphation at C6 but this occurs more frequently on GlcNAc than D-Gal. Sulphation along the developing KS chain is not uniform and contains regions of variable length where no sulphation occurs, regions which are monosulphated mainly on GlcNAc and further regions of high sulphation where both of the repeat disaccharides are sulphated. Each of these respective regions in the KS chain can be of variable length leading to KS complexity in terms of chain length and charge localization along the KS chain. Like other GAGs, it is these variably sulphated regions in KS which define its interactive properties with ligands such as growth factors, morphogens and cytokines and which determine the functional properties of tissues containing KS. Further adding to KS complexity is the identification of three different linkage structures in KS to asparagine (N-linked) or to threonine or serine residues (O-linked) in proteoglycan core proteins which has allowed the categorization of KS into three types, namely KS-I (corneal KS, N-linked), KS-II (skeletal KS, O-linked) or KS-III (brain KS, O-linked). KS-I to -III are also subject to variable addition of L-fucose and sialic acid groups. Furthermore, the GlcNAc residues of some members of the mucin-like glycoprotein family can also act as acceptor molecules for the addition of D-Gal and GlcNAc residues which can also be sulphated leading to small low sulphation glycoforms of KS. These differ from the more heavily sulphated KS chains found on proteoglycans. Like other GAGs, KS has evolved molecular recognition and information transfer properties over hundreds of millions of years of vertebrate and invertebrate evolution which equips them with cell mediatory properties in normal cellular processes and in aberrant pathological situations such as in tumourogenesis. Two KS-proteoglycans in particular, podocalyxin and lumican, are cell membrane, intracellular or stromal tissue–associated components with roles in the promotion or regulation of tumour development, mucin-like KS glycoproteins may also contribute to tumourogenesis. A greater understanding of the biology of KS may allow better methodology to be developed to more effectively combat tumourogenic processes