The homeobox gene TGIF1 is required for chicken ovarian cortical development and generation of the juxtacortical medulla.

During early embryogenesis in amniotic vertebrates, the gonads differentiate into either ovaries or testes. The first cell lineage to differentiate gives rise to the supporting cells: Sertoli cells in males and pre-granulosa cells in females. These key cell types direct the differentiation of the other cell types in the gonad, including steroidogenic cells. The gonadal surface epithelium and the interstitial cell populations are less well studied, and little is known about their sexual differentiation programs. Here, we show the requirement of the homeobox transcription factor gene TGIF1 for ovarian development in the chicken embryo. TGIF1 is expressed in the two principal ovarian somatic cell populations: the cortex and the pre-granulosa cells of the medulla. TGIF1 expression is associated with an ovarian phenotype in estrogen-mediated sex reversal experiments. Targeted misexpression and gene knockdown indicate that TGIF1 is required, but not sufficient, for proper ovarian cortex formation. In addition, TGIF1 is identified as the first known regulator of juxtacortical medulla development. These findings provide new insights into chicken ovarian differentiation and development, specifically cortical and juxtacortical medulla formation.

Forecasting stroke-like episodes and outcomes in mitochondrial disease.

In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors. Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk. The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82-0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005]. Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.

CRISPR mediated somatic cell genome engineering in the chicken.

Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model.

NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

APELIN promotes hematopoiesis from human embryonic stem cells.

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR mAb, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs, and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore, APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells.

Long-term outcome of paediatric-onset multiple sclerosis: a population-based study.

BACKGROUND: Age of onset of multiple sclerosis (MS) peaks in the 3rd and 4th decades and is rarely less than 18. Robust longitudinal studies in paediatric-onset MS (POMS) are limited, and a clearer understanding of outcome could optimise management strategies. METHODS: Patients with disease onset <18 years were identified from a prospective population-based register. Clinical features including presenting symptoms, time to Expanded Disability Status Scale (EDSS) 4.0, 6.0 and 8.0 and onset of secondary progression were compared with patients with adult-onset MS (AOMS). RESULTS: 111 POMS patients were identified from a cohort of 2068. No significant differences in sex ratio, familial recurrence, relapse rate, ethnicity or clinical symptoms at presentation were identified between POMS and AOMS. However, interval to second relapse was longer (5 vs 2.6 years, p=0.04) and primary progressive disease was less common (0.9% vs 8.5%, p=0.003) in POMS than in AOMS. POMS patients also took longer to develop secondary progressive disease (32 vs 18 years, p=0.0001) and to reach disability milestones (EDSS 4.0, 23.8 vs 15.5 years, p<0.0001; EDSS 6.0, 30.8 vs 20.4 years, p<0.0001; EDSS 8.0, 44.7 vs 39 years, p=0.02), but did so between 7.0 and 12 years younger than in AOMS. CONCLUSIONS: 5.4% of patients with MS have POMS (2.7% <16 years; 0.3% <10 years) and have distinct phenotypic characteristics in early disease. Furthermore, while patients with POMS take longer to reach disability milestones, they do so at a younger age than their adult counterparts and could be considered to have a poorer prognosis. Management strategies for these patients should take account of these data.

Temporal evolution of remission following multiple sclerosis relapse and predictors of outcome.

BACKGROUND: Relapse is a characteristic clinical feature of multiple sclerosis (MS) and is commonly employed as a measure of efficacy following therapeutic intervention. However, less is known about the temporal evolution of subsequent disability or factors predicting recovery. OBJECTIVES: The objective of this study was to assess the pattern of recovery following relapse and identify factors which predict recovery and residual disability following relapse. METHODS: A total of 226 relapses were studied prospectively in a cohort of 144 patients with standardised clinical assessments of physical disability including Expanded Disability Status Scale (EDSS), 10-m timed walk, 9-hole peg test and Multiple Sclerosis Impact Scale (MSIS-29) at 0, 2, 6 and 12 months. A total of 82 patients completed 12 months of follow up without further relapse. RESULTS: Thirty per cent of relapses were severe (change in EDSS >2.0) of which 11% failed to recover. All measures showed significant improvement at 2 months but additional improvement was also observed in 9-hole peg test and MSIS-29 up to 12 months following initial assessment. Mean time to second relapse was 382 days. The only predictor of relapse severity in the model tested was younger age; however, increasing age and initial relapse severity were also predictors of poor outcome. CONCLUSIONS: This study shows that the majority of improvement in physical disability following relapse occurs by 2 months but that more subtle recovery can take place over 12 months in a small sub-group of patients. These data will aid in patient counselling and will also inform the timing of therapeutic intervention and physical support.

Prion-like domains drive CIZ1 assembly formation at the inactive X chromosome.

CIZ1 forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner and is required for accurate maintenance of polycomb targets genome-wide. Here we address requirements for assembly formation and show that CIZ1 undergoes two direct interactions with Xist, via independent N- and C-terminal domains. Interaction with Xist, assembly at Xi, and complexity of self-assemblies formed in vitro are modulated by two alternatively spliced glutamine-rich prion-like domains (PLD1 and 2). PLD2 is dispensable for accumulation at existing CIZ1-Xi assemblies in wild-type cells but is required in CIZ1-null cells where targeting, assembly, and enrichment for H3K27me3 and H2AK119ub occur de novo. In contrast, PLD1 is required for both de novo assembly and accumulation at preexisting assemblies and, in vitro, drives formation of a stable fibrillar network. Together they impart affinity for RNA and a complex relationship with repeat E of Xist. These data show that alternative splicing of two PLDs modulates CIZ1's ability to build large RNA-protein assemblies.

Complement regulator factor H as a serum biomarker of multiple sclerosis disease state.

Multiple sclerosis has a variable phenotypic presentation and subsequent disease course that, although unpredictable at disease onset, is of crucial importance in guiding interventions. Effective and accessible biomarkers are required in order to stratify patients and inform treatment. We examined whether the complement regulator factor H and its Tyr402His polymorphism, recently implicated as biomarkers in other chronic inflammatory central nervous system conditions, might identify or predict specific pathological processes and outcomes in multiple sclerosis. Employing novel assays, we measured factor H and its His402 variant in serum from 350 patients with multiple sclerosis classified according to disease course and relapse status. Serum factor H levels were significantly higher in progressive disease (P < 0.001) compared to controls and relapsing patients, after controlling for variables including disease duration, age, gender, disability and treatment. Serum factor H levels were capable of distinguishing secondary progressive from relapsing remitting disease (excluding patients in clinical relapse) with a sensitivity of 89.41%, specificity of 69.47% and a positive predictive value of 72.38%. Acute relapse was also associated with transiently increased factor H levels (P = 0.009) compared to stable relapsing disease. In clinically stable patients, factor H levels remained constant over 1 year (coefficient of variation percentage = 6.8), however, in patients in transition from relapsing to progressive disease, factor H levels significantly increased over a period of 2 years (P = 0.007). Concentration of the His402 variant in heterozytgotes was significantly higher in secondary progressive (P < 0.01) and primary progressive (P < 0.05) disease, suggesting altered expression or consumption of variants when factor H is upregulated. Serum factor H may be an effective indicator of progression and a practical and accessible biomarker and stratifying tool in determining disease course, providing objective evidence to help guide therapeutic decisions.

Enforced expression of Mixl1 during mouse ES cell differentiation suppresses hematopoietic mesoderm and promotes endoderm formation.

The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1(C) ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)(+) cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1(C) ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1(C) EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation.

Adhesion G-protein-coupled receptor, GPR56, is required for Müllerian duct development in the chick.

The embryonic Müllerian ducts give rise to the female reproductive tract (fallopian tubes, uterus and upper vagina in humans, the oviducts in birds). Embryonic Müllerian ducts initially develop in both sexes, but later regress in males under the influence of anti-Müllerian hormone. While the molecular and endocrine control of duct regression in males have been well studied, early development of the ducts in both sexes is less well understood. Here, we describe a novel role for the adhesion G protein-coupled receptor, GPR56, in development of the Müllerian ducts in the chicken embryo. GPR56 is expressed in the ducts of both sexes from early stages. The mRNA is present during the elongation phase of duct formation, and it is restricted to the inner Müllerian duct epithelium. The putative ligand, Collagen III, is abundantly expressed in the Müllerian duct at the same developmental stages. Knockdown of GPR56 expression using in ovo electroporation results in variably truncated ducts, with a loss of expression of both epithelial and mesenchymal markers of duct development. Over-expression of GPR56 in vitro results in enhanced cell proliferation and cell migration. These results show that GPR56 plays an essential role in avian Müllerian duct development through the regulation of duct elongation.

Insights into Gonadal Sex Differentiation Provided by Single-Cell Transcriptomics in the Chicken Embryo.

Although the genetic triggers for gonadal sex differentiation vary across species, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using single-cell sequencing in the embryonic chicken gonad during sexual differentiation. The data show that chicken embryonic-supporting cells do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, they derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes and derivation of embryonic steroidogenic cells from a differentiated supporting-cell lineage. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.

Feasibility study of early outpatient review and early cardiac rehabilitation after cardiac surgery: mixed-methods research design-a study protocol.

INTRODUCTION: Following cardiac surgery, patients currently attend an outpatient review 6 weeks after hospital discharge, where recovery is assessed and suitability to commence cardiac rehabilitation (CR) is determined. CR is then started from 8 weeks. Following a median sternotomy, cardiac surgery patients are required to refrain from upper body exercises, lifting of heavy objects and other strenuous activities for 12 weeks. A delay in starting CR can prolong the recovery process, increase dependence on family/carers and can cause frustration. However, current guidelines for activity and exercise after median sternotomy have been described as restrictive, anecdotal and increasingly at odds with modern clinical guidance for CR. This study aims to examine the feasibility of bringing forward outpatient review and starting CR earlier. METHODS AND ANALYSES: This is a multicentre, randomised controlled, open feasibility trial comparing postoperative outpatient review 6 weeks after hospital discharge, followed by CR commencement from 8 weeks (control arm) versus, postoperative outpatient review 3 weeks after hospital discharge, followed by commencement of CR from 4 weeks (intervention arm). The study aims to recruit 100 eligible patients, aged 18-80 years who have undergone elective or urgent cardiac surgery involving a full median sternotomy, over a 7-month period across two centres. Feasibility will be measured by consent, recruitment, retention rates and attendance at appointments and CR sessions. Qualitative interviews with trial participants and staff will explore issues around study processes and acceptability of the intervention and the findings integrated with the feasibility trial outcomes to inform the design of a future full-scale randomised controlled trial. ETHICS AND DISSEMINATION: Ethics approval was granted by East Midlands-Derby Research Ethics Committee on 10 January 2019. The findings will be presented at relevant conferences disseminated via peer-reviewed research publications, and to relevant stakeholders. TRIAL REGISTRATION NUMBER: ISRCTN80441309.

Contribution of relapses to disability in multiple sclerosis.

The impact of relapses on long-term disability in multiple sclerosis remains unclear; however some evidence suggests that relapses play an important role in determining subsequent prognosis. We report on outcome, prognostic factors for recovery and the contribution of relapses to the accumulation of fixed disability in a large series of patients with documented relapses. Two hundred and seventy-nine relapses in 182 patients were assessed before, during and after relapse by expanded disability status scale and data analysed to assess degree of recovery. Factors affecting outcome were considered including sex, age and site of relapse. Mean EDSS prior to relapse was 3.73, during relapse 5.18 and post relapse 4.23. Mean interval to post relapse assessment was 127 days post relapse. Mean residual change in EDSS score (pre to post relapse) was 0.50 points. Overall 49.4 % of patients had a residual increase in disability post relapse of at least 0.5 EDSS points and 32.7 % had an increase of at least 1 point. No significant difference was observed in mean residual EDSS for sex, site of relapse or age at relapse or in the proportion of patients with a residual increase in disability of > or = 1 EDSS point post relapse. 14.4 % of patients had no increase in EDSS score during relapse compared to pre relapse. These results suggest that acute relapses are commonly associated with an objective worsening of disability in the majority of patients with MS and that recovery is incomplete in approximately half and not influenced by gender, age or site of lesion. Therapies which reduce relapse frequency and/or severity might therefore be expected to slow or prevent worsening of disability if initiated prior to the onset of more permanent damage.

Sex determination and gonadal sex differentiation in the chicken model.

Our understanding of avian sex determination and gonadal development is derived primarily from the studies in the chicken. Analysis of gynandromorphic chickens and experimental chimeras indicate that sexual phenotype is at least partly cell autonomous in the chicken, with sexually dimorphic gene expression occurring in different tissue and different stages. Gonadal sex differentiation is just one of the many manifestations of sexual phenotype. As in other birds, the chicken has a ZZ male: ZW female sex chromosome system, in which the male is the homogametic sex. Most evidence favours a Z chromosome dosage mechanism underling chicken sex determination, with little evidence of a role for the W chromosome. Indeed, the W appears to harbour a small number of genes that are un-related to sexual development, but have been retained because they are dosage sensitive factors. As global Z dosage compensation is absent in birds, Z-linked genes may direct sexual development in different tissues (males having on average 1.5 to 2 times the expression level of females). In the embryonic gonads, the Z-linked DMRT1 gene plays a key role in testis development. Beyond the gonads, other combinations of Z-linked genes may govern sexual development, together with a role for sex steroid hormones. Gonadal DMRT1 is thought to activate other players in testis development, namely SOX9 and AMH, and the recently identified HEMGN gene. DMRT1 also represses ovarian pathway genes, such as FOXL2 and CYP19A1. A lower level of DMRT1 expression in the female gonads is compatible with activation of the ovarian pathway. Some outstanding questions include how the key testis and ovary genes, DMRT1 and FOXL2, are regulated. In addition, confirmation of the central role of these genes awaits genome editing approaches.

Gonadal and Endocrine Analysis of a Gynandromorphic Chicken.

Birds have a ZZ male and ZW female sex chromosome system. The relative roles of genetics and hormones in regulating avian sexual development have been revealed by studies on gynandromorphs. Gynandromorphs are rare bilateral sex chimeras, male on one side of the body and female on the other. We examined a naturally occurring gynandromorphic chicken that was externally male on the right side of the body and female on the left. The bird was diploid but with a mix of ZZ and ZW cells that correlated with the asymmetric sexual phenotype. The male side was 96% ZZ, and the female side was 77% ZZ and 23% ZW. The gonads of this bird at sexual maturity were largely testicular. The right gonad was a testis, with SOX9+ Sertoli cells, DMRT1+ germ cells, and active spermatogenesis. The left gonad was primarily testicular, but with some peripheral aromatase-expressing follicles. The bird had low levels of serum estradiol and high levels of testosterone, as expected for a male. Despite the low percentage of ZW cells on that side, the left side had female sex-linked feathering, smaller muscle mass, smaller leg and spur, and smaller wattle than the male side. This indicates that these sexually dimorphic structures must be at least partly independent of sex steroid effects. Even a small percentage of ZW cells appears sufficient to support female sexual differentiation. Given the lack of chromosome-wide dosage compensation in birds, various sexually dimorphic features may arise due to Z-gene dosage differences between the sexes.

Genetic Manipulation of the Avian Urogenital System Using In Ovo Electroporation.

One of the advantages of the avian embryo as an experimental model is its in ovo development and hence accessibility for genetic manipulation. Electroporation has been used extensively in the past to study gene function in chicken and quail embryos . Readily accessible tissues such as the neural tube, somites, and limb bud, in particular, have been targeted. However, more inaccessible tissues, such as the embryonic urogenital system , have proven more challenging to study. Here, we describe the use of in ovo electroporation of TOL2 vectors or RCASBP avian viral vectors for the rapid functional analysis of genes involved in avian sex determination and urogenital development . In the context of the developing urogenital system , these vectors have inherent advantages and disadvantages, which will be considered here. Either vector can both be used for mis-expressing a gene and for targeting endogenous gene knockdown via expression of short hairpin RNAs (shRNAs). Both of these vectors integrate into the genome and are hence spread throughout developing tissues. Going forward, electroporation could be combined with CRISPR/Cas9 technology for targeted genome editing in the avian urogenital system .