Integrated analysis of Wnt signalling system component gene expression.

Wnt signalling controls patterning and differentiation across many tissues and organs of the developing embryo through temporally and spatially restricted expression of multi-gene families encoding ligands, receptors, pathway modulators and intracellular components. Here, we report an integrated analysis of key genes in the 3D space of the mouse embryo across multiple stages of development. We applied a method for 3D/3D image transformation to map all gene expression patterns to a single reference embryo for each stage, providing both visual analysis and volumetric mapping allowing computational methods to interrogate the combined expression patterns. We identify territories where multiple Wnt and Fzd genes are co-expressed and cross-compare all patterns, including all seven Wnt paralogous gene pairs. The comprehensive analysis revealed regions in the embryo where no Wnt or Fzd gene expression is detected, and where single Wnt genes are uniquely expressed. This work provides insight into a previously unappreciated level of organisation of expression patterns, as well as presenting a resource that can be utilised further by the research community for whole-system analysis.

Building a Co-ordinated Musculoskeletal System: The Plasticity of the Developing Skeleton in Response to Muscle Contractions.

The skeletal musculature and the cartilage, bone and other connective tissues of the skeleton are intimately co-ordinated. The shape, size and structure of each bone in the body is sculpted through dynamic physical stimuli generated by muscle contraction, from early development, with onset of the first embryo movements, and through repair and remodelling in later life. The importance of muscle movement during development is shown by congenital abnormalities where infants that experience reduced movement in the uterus present a sequence of skeletal issues including temporary brittle bones and joint dysplasia. A variety of animal models, utilising different immobilisation scenarios, have demonstrated the precise timing and events that are dependent on mechanical stimulation from movement. This chapter lays out the evidence for skeletal system dependence on muscle movement, gleaned largely from mouse and chick immobilised embryos, showing the many aspects of skeletal development affected. Effects are seen in joint development, ossification, the size and shape of skeletal rudiments and tendons, including compromised mechanical function. The enormous plasticity of the skeletal system in response to muscle contraction is a key factor in building a responsive, functional system. Insights from this work have implications for our understanding of morphological evolution, particularly the challenging concept of emergence of new structures. It is also providing insight for the potential of physical therapy for infants suffering the effects of reduced uterine movement and is enhancing our understanding of the cellular and molecular mechanisms involved in skeletal tissue differentiation, with potential for informing regenerative therapies.

Developing a health promoting university in Trinity College Dublin-overview and outline process evaluation.

Higher Education Institutions (HEIs) have the potential to impact positively on the health and wellbeing of their staff and students. Using and expanding on the 'health promoting university' (HPU) platform within HEIs, this article provides a description of 'Healthy Trinity', which is an initiative underway in Trinity College Dublin, the University of Dublin. First, Healthy Trinity is contextualized in background literature including international and national policy and practice. Second, an overview of Healthy Trinity is provided including its vision and goals. Third, the article describes the steps taken relating to the identification of stakeholders and use of a network and a co-lead model. Within this approach, the article describes a partnership approach whereby responsibilities regarding health and wellbeing are shared by individuals and the institution. Fourth, the design and implementation of Healthy Trinity is discussed by taking a 'settings approach', in which the emphasis for change is placed on individual behaviours, environment, policy and organizational culture. Consideration is given to the interplay between intervention, implementation strategy and context for successful systemic implementation. The fifth element presented is the early-stage challenges encountered during implementation, such as the need to secure recurrent funding and the importance of having a direct input to the governance of the University to enable systemic change. The sixth and final component of the article is an outline of Healthy Trinity's intention to utilize a process evaluation of the early implementation phases of this complex intervention within a settings approach. Potential deliverables and impacts of this HPU initiative are presented and discussed.

Universities, such as Trinity College Dublin, the University of Dublin, can be looked at as a community of staff and students. The university community has needs in terms of health and wellbeing. 'Healthy Trinity' attempted to build strategies and practices to meet these needs for its community. The approach taken was from multiple angles and involved students and staff, focusing on both individual and organizational responsibility to promote and encourage healthy behaviours. Healthy Trinity achieved some successes as well as encountering some challenges. This article explores how the university might build upon the successes of Healthy Trinity in order to embed a culture which prioritizes health and wellbeing for the entire university community. The article also looks at the broader impact of achieving this goal, namely the University's contribution to a healthier community beyond the university setting.

Geometric analysis of chondrogenic self-organisation of embryonic limb bud cells in micromass culture.

Spatial and temporal control of chondrogenesis generates precise, species-specific patterns of skeletal structures in the developing vertebrate limb. The pattern-template is laid down when mesenchymal cells at the core of the early limb bud condense and undergo chondrogenic differentiation. Although the mechanisms involved in organising such complex patterns are not fully understood, the interplay between BMP and Wnt signalling pathways is fundamental. Primary embryonic limb bud cells grown under high-density micromass culture conditions spontaneously create a simple cartilage nodule pattern, presenting a model to investigate pattern generation. We describe a novel analytical approach to quantify geometric properties and spatial relationships between chondrogenic condensations, utilizing the micromass model. We follow the emergence of pattern in live cultures with nodules forming at regular distances, growing and changing shape over time. Gene expression profiling supports rapid chondrogenesis and transition to hypertrophy, mimicking the process of endochondral ossification within the limb bud. Manipulating the signalling environment through addition of BMP or Wnt ligands, as well as the BMP pathway antagonist Noggin, altered the differentiation profile and nodule pattern. BMP2 addition increased chondrogenesis while WNT3A or Noggin had the opposite effect, but with distinct pattern outcomes. Titrating these pro- and anti-chondrogenic factors and examining the resulting patterns support the hypothesis that regularly spaced cartilage nodules formed by primary limb bud cells in micromass culture are influenced by the balance of Wnt and BMP signalling under a Turing-like mechanism. This study demonstrates an approach for investigating the mechanisms governing chondrogenic spatial organization using simple micromass culture.

Precise spatial restriction of BMP signaling in developing joints is perturbed upon loss of embryo movement.

Dynamic mechanical loading of synovial joints is necessary for normal joint development, as evidenced in certain clinical conditions, congenital disorders and animal models where dynamic muscle contractions are reduced or absent. Although the importance of mechanical forces on joint development is unequivocal, little is known about the molecular mechanisms involved. Here, using chick and mouse embryos, we observed that molecular changes in expression of multiple genes analyzed in the absence of mechanical stimulation are consistent across species. Our results suggest that abnormal joint development in immobilized embryos involves inappropriate regulation of Wnt and BMP signaling during definition of the emerging joint territories, i.e. reduced ß-catenin activation and concomitant upregulation of pSMAD1/5/8 signaling. Moreover, dynamic mechanical loading of the developing knee joint activates Smurf1 expression; our data suggest that Smurf1 insulates the joint region from pSMAD1/5/8 signaling and is essential for maintenance of joint progenitor cell fate.

Localization of YAP activity in developing skeletal rudiments is responsive to mechanical stimulation.

BACKGROUND: Normal skeletal development, in particular ossification, joint formation and shape features of condyles, depends on appropriate mechanical input from embryonic movement but it is unknown how such physical stimuli are transduced to alter gene regulation. Hippo/Yes-Associated Protein (YAP) signalling has been shown to respond to the physical environment of the cell and here we specifically investigate the YAP effector of the pathway as a potential mechanoresponsive mediator in the developing limb skeleton. RESULTS: We show spatial localization of YAP protein and of pathway target gene expression within developing skeletal rudiments where predicted biophysical stimuli patterns and shape are affected in immobilization models, coincident with the period of sensitivity to movement, but not coincident with the expression of the Hippo receptor Fat4. Furthermore, we show that under reduced mechanical stimulation, in immobile, muscle-less mouse embryos, this spatial localization is lost. In culture blocking YAP reduces chondrogenesis but the effect differs depending on the timing and/or level of YAP reduction. CONCLUSIONS: These findings implicate YAP signalling, independent of Fat4, in the transduction of mechanical signals during key stages of skeletal patterning in the developing limb, in particular endochondral ossification and shape emergence, as well as patterning of tissues at the developing synovial joint.

Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure.

Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.

Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis.

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3ß (Ser9), nuclear accumulation of ß-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-ß (TGF-ß); Wnt6 reversed TGF-ß-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-ß-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/ß(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.

Notochord manipulation does not impact oesophageal and tracheal formation from isolated foregut in 3D explant culture.

BACKGROUND: Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of notochord signaling on the adjacent foregut is not known. The purpose of this study was to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. METHODS: Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48 h with native notochord intact (n = 6), notochord removed (n = 10) or additional notochord transplanted from stage matched controls (n = 8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. RESULTS: Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord. In specimens with additional notochord transplants, foregut morphology and molecular patterning were comparable to controls whether or not the native notochord was maintained. In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. CONCLUSION: The relationship between the embryonic foregut and notochord is complex and ill-defined; however, the notochord does not contribute essentially to oesophagus and trachea formation beyond E9 in the mouse, and the transplantation of additional notochord does not disrupt foregut separation in 3D explant culture.

Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways.

BACKGROUND: Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. RESULTS: We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response. CONCLUSIONS: This work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. In particular it highlights the Wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response.

Altered Tbx1 gene expression is associated with abnormal oesophageal development in the adriamycin mouse model of oesophageal atresia/tracheo-oesophageal fistula.

INTRODUCTION: Oesophageal atresia/tracheo-oesophageal atresia (OA/TOF) frequently arises with associated anomalies and has been clinically linked with 22q11 deletion syndromes, a group of conditions due to Tbx1 gene mutation which include Di George syndrome. Tbx1 and Tbx2 genes modulate pharyngeal and cardiac development, but are also expressed in the developing foregut and are known to interact with key signalling pathways described in oesophageal formation including bone morphogenic proteins. The adriamycin mouse model (AMM) reliably displays OA/TOF-like foregut malformations providing a powerful system for investigating the disturbances in gene regulation and morphology involved in tracheo-oesophageal malformations. We hypothesised that foregut abnormalities observed in the AMM are associated with altered Tbx1 and Tbx2 gene expression. METHODS: Time-mated CBA/Ca mice received intra-peritoneal injection of adriamycin (for treated) or saline (for controls) on embryonic days (E)7 and 8. Untreated Cd1 embryos were used to establish normal expression patterns. Embryos harvested on E9-E11 underwent whole-mount in situ hybridization with labelled RNA probes for Tbx1 and Tbx2. Optical projection tomography was used to visualise expression in whole embryos by 3D imaging. RESULTS: Tbx1 expression was visualised in a highly specific pattern in the proximal oesophageal endoderm in normal and control embryos. In the AMM, extensive ectopic expression of Tbx1 was detected in the dorsal foregut and adjacent to the TOF. The focally restricted oesophageal expression pattern persisted in the AMM, but was posteriorly displaced in relation to the tracheal bifurcation. Tbx2 was widely expressed in the ventral foregut mesoderm of controls, lacking specific endoderm localisation. In the AMM, altered Tbx2 expression in the foregut was only seen in severely affected embryos. CONCLUSION: Highly specific Tbx1 expression in the proximal oesophageal endoderm suggests that Tbx1 may be an important regulator of normal oesophageal development. Altered Tbx1 expression in dorsal foregut and adjacent to the TOF in the AMM suggests that Tbx1 gene disruption may contribute to the pathogenesis of tracheo-oesophageal malformations.

Timeliness of second opinion appointed doctors' assessments of treatment plans for patients detained in medium security hospitals in London, UK.

BACKGROUND: Mental health legislation in England & Wales requires assessment by a second opinion appointed doctor (SOAD) to safeguard the rights of patients detained in a hospital under this law if they are either refusing certain treatments or are deemed incapable of consenting to them. AIMS: Our aim was to evaluate timeliness of SOAD assessments. METHOD: Data were collected from the Mental Health Act administrator on all SOAD requests in 1 year for all in-patients in two English medium security hospital units. RESULTS: One hundred and six patients required a SOAD assessment during the 1-year period examined, of a combined resident total of 295, as did a further 14 patients who had been discharged from hospital and were subject to a community treatment order. About half of the inpatients were seen by a SOAD within 30 days and a further quarter within 60 days, but the remaining quarter waited up to 150 days or more. DISCUSSION AND IMPLICATIONS: These results suggest that in these trusts, unlikely to be atypical, neither patients nor clinicians are being adequately protected by legal safeguards on decisions to treat with medication (or electroconvulsive therapy) in the event of impaired competence for decisions about mental health treatments. There should be clear standards for the appropriate length of time from referral to assessment by an independent doctor (SOAD). Compliance with standards should be transparent, so anonymised data on the matter should be routinely collected and stored by health trust Mental Health Act offices. Data should be monitored at agreed intervals by an independent body.

Do some patients receive unnecessary parenteral nutrition after pancreatoduodenectomy? Results from an international multicentre study.

Backgrounds/Aims: After pancreatoduodenectomy (PD), an early oral diet is recommended; however, the postoperative nutritional management of PD patients is known to be highly variable, with some centers still routinely providing parenteral nutrition (PN). Some patients who receive PN experience clinically significant complications, underscoring its judicious use. Using a large cohort, this study aimed to determine the proportion of PD patients who received postoperative nutritional support (NS), describe the nature of this support, and investigate whether receiving PN correlated with adverse perioperative outcomes. Methods: Data were extracted from the Recurrence After Whipple's study, a retrospective multicenter study of PD outcomes. Results: In total, 1,323 patients (89%) had data on their postoperative NS status available. Of these, 45% received postoperative NS, which was "enteral only," "parenteral only," and "enteral and parenteral" in 44%, 35%, and 21% of cases, respectively. Body mass index < 18.5 kg/m2 (p = 0.03), absence of preoperative biliary stenting (p = 0.009), and serum albumin < 36 g/L (p = 0.009) all correlated with receiving postoperative NS. Among those who did not develop a serious postoperative complication, i.e., those who had a relatively uneventful recovery, 20% received PN. Conclusions: A considerable number of patients who had an uneventful recovery received PN. PN is not without risk, and should be reserved for those who are unable to take an oral diet. PD patients should undergo pre- and postoperative assessment by nutrition professionals to ensure they are managed appropriately, and to optimize perioperative outcomes.

eChickAtlas: an introduction to the database.

The precise control of gene expression is critical in embryonic development. Quantitative assays, such as microarrays and RNA sequencing, provide gene expression levels for a large number of genes, but do not contain spatial information. In contrast, in situ methods, such as in situ hybridization and immunohistochemistry, provide spatial resolution, but poor quantification and can only reveal the expression of one, or very few genes at a time. Furthermore, the usual methods of documenting the results, by photographing whole mounts or sections, makes it very difficult to assess the three-dimensional (3D) relationships between expressing and nonexpressing cells. Optical projection tomography (OPT) can capture the full 3D expression pattern in a whole embryo at a reasonable level of resolution and at moderately high throughput. A large database containing spatio-temporal patterns of expression for the mouse (e-Mouse Atlas Project, EMAP, www.emouseatlas.org) has been created, incorporating 3D information. Like the mouse, the chick is an important model in developmental biology and translational studies. To facilitate comparisons between these important model organisms, we have created a 3D anatomical atlas, accompanied by an anatomical ontology of the chick embryo and a database of gene expression patterns during chick development. This database is publicly available (www.echickatlas.org).

Renal hypertension: an unusual cause for a common problem.

A 9-year-old girl with longstanding headaches presented acutely with rash, which disappeared quickly on treatment with oral phenoxymethylpenicillin. It was attributed to streptococcal infection as group A streptococcus was isolated from throat swab. She was incidentally found to have high blood pressure on routine screening on admission. Subsequently, 'fibromuscular dysplasia' was confirmed on renal angiogram, which showed a characteristic beaded appearance. It is a good clinical practice to check blood pressure in any child seen for whatever reason.

Mechanical regulation of skeletal development.

Development of the various components of a normal skeleton requires highly regulated signalling systems that co-ordinate spatial and temporal patterns of cell division, cell differentiation, and morphogenesis. Much work in recent decades has revealed cascades of molecular signalling, acting through key transcription factors to regulate, for example, organized chondrogenic and osteogenic differentiation. It is now clear that mechanical stimuli are also required for aspects of skeletogenesis but very little is known about how the mechanical signals are integrated with classic biochemical signalling. Spatially organized differentiation is vital to the production of functionally appropriate tissues contributing to precise, region specific morphologies, for example transient chondrogenesis of long bone skeletal rudiments, which prefigures osteogenic replacement of the cartilage template, compared with the production of permanent cartilage at the sites of articulation. Currently a lack of understanding of how these tissues are differentially regulated hampers efforts to specifically regenerate stable bone and cartilage. Here, we review current research revealing the influence of mechanical stimuli on specific aspects of skeletal development and refer to other developing systems to set the scene for current and future work to uncover the molecular mechanisms involved. We integrate this with a brief overview of the effects of mechanical stimulation on stem cells in culture bringing together developmental and tissue engineering aspects of mechanoregulation of cell behavior. A better understanding of the molecular mechanisms that link mechanical stimuli to transcriptional control guiding cell differentiation will lead to new ideas about how to effectively prime stem cells for tissue engineering and regenerative therapies.

Primary care and youth mental health in Ireland: qualitative study in deprived urban areas.

BACKGROUND: Mental disorders account for six of the 20 leading causes of disability worldwide with a very high prevalence of psychiatric morbidity in youth aged 15-24 years. However, healthcare professionals are faced with many challenges in the identification and treatment of mental and substance use disorders in young people (e.g. young people's unwillingness to seek help from healthcare professionals, lack of training, limited resources etc.) The challenge of youth mental health for primary care is especially evident in urban deprived areas, where rates of and risk factors for mental health problems are especially common. There is an emerging consensus that primary care is well placed to address mental and substance use disorders in young people especially in deprived urban areas. This study aims to describe healthcare professionals' experience and attitudes towards screening and early intervention for mental and substance use disorders among young people (16-25 years) in primary care in deprived urban settings in Ireland. METHODS: The chosen method for this qualitative study was inductive thematic analysis which involved semi-structured interviews with 37 healthcare professionals from primary care, secondary care and community agencies at two deprived urban centres. RESULTS: We identified three themes in respect of interventions to increase screening and treatment: (1) Identification is optimised by a range of strategies, including raising awareness, training, more systematic and formalised assessment, and youth-friendly practices (e.g. communication skills, ensuring confidentiality); (2) Treatment is enhanced by closer inter-agency collaboration and training for all healthcare professionals working in primary care; (3) Ongoing engagement is enhanced by motivational work with young people, setting achievable treatment goals, supporting transition between child and adult mental health services and recognising primary care's longitudinal nature as a key asset in promoting treatment engagement. CONCLUSIONS: Especially in deprived areas, primary care is central to early intervention for youth mental health. Identification, treatment and continuing engagement are likely to be enhanced by a range of strategies with young people, healthcare professionals and systems. Further research on youth mental health and primary care, including qualitative accounts of young people's experience and developing complex interventions that promote early intervention are priorities.

Effect of abnormal notochord delamination on hindgut development in the Adriamycin mouse model.

BACKGROUND: Adriamycin mouse model (AMM) is a model of VACTERL anomalies. Sonic hedgehog (Shh) pathway, sourced by the notochord, is implicated of anorectal malformations. We hypothesized hindgut anomalies observed in the AMM are the result of abnormal effect of the notochord. METHODS: Time-mated CBA/Ca mice received two intraperitoneal injections of Adriamycin (6 mg/kg) or saline as control on embryonic day (E) 7 and 8. Fetuses were harvested from E9 to E11, stained following whole mount in situ hybridization with labeled RNA probes to detect Shh and Fork head box F1(Foxf1) transcripts. Immunolocalization with endoderm marker Hnf3ß was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of expressions. RESULTS: In AMM, the notochord was abnormally displaced ventrally with attachment to the hindgut endoderm in 71 % of the specimens. In 32 % of the treated embryos abnormal hindgut ended blindly in a cystic structure, and both of types were remarked in 29 % of treated embryos. Endodermal Shh and mesenchymal Foxf1 genes expression were preserved around the hindgut cystic malformation. CONCLUSIONS: The delamination of the developing notochord in the AMM is disrupted, which may influence signaling mechanisms from the notochord to the hindgut resulting in abnormal patterning of the hindgut.

Differential deployment of paralogous Wnt genes in the mouse and chick embryo during development.

Genes encoding Wnt ligands are crucial in body patterning and are highly conserved among metazoans. Given their conservation at the protein-coding level, it is likely that changes in where and when these genes are active are important in generating evolutionary variations. However, we lack detailed knowledge about how their deployment has diverged. Here, we focus on four Wnt subfamilies (Wnt2, Wnt5, Wnt7, and Wnt8) in mammalian and avian species, consisting of a paralogous gene pair in each, believed to have duplicated in the last common ancestor of vertebrates. We use three-dimensional imaging to capture expression patterns in detail and carry out systematic comparisons. We find evidence of greater divergence between these subgroup paralogues than the respective orthologues, consistent with some level of subfunctionalization/neofunctionalization in the common vertebrate ancestor that has been conserved. However, there were exceptions; in the case of chick Wnt2b, individual sites were shared with both mouse Wnt2 and Wnt2b. We also find greater divergence, between paralogues and orthologues, in some subfamilies (Wnt2 and Wnt8) compared to others (Wnt5 and Wnt7) with the more highly similar expression patterns showing more extensive expression in more structures in the embryo. Wnt8 genes were most restricted and most divergent. Major sites of expression for all subfamilies include CNS, limbs, and facial region, and in general there were more similarities in gene deployment in these territories with divergent patterns featuring more in organs such as heart and gut. A detailed comparison of gene expression patterns in the limb showed similarities in overall combined domains across species with notable differences that may relate to lineage-specific morphogenesis.

Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells.

Hydrostatic pressure (HP) is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-ß3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs), synovial membrane derived stem cells (SDSCs) and infrapatellar fat pad derived stem cells (FPSCs) were subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-ß3 (0, 1 and 10 ng/mL) for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-ß3 stimulation. HP in the absence of TGF-ß3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-ß3 (1 ng/mL), HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-ß3 (10 ng/mL). Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.