DMRT1-mediated regulation of TOX3 modulates expansion of the gonadal steroidogenic cell lineage in the chicken embryo.

During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data have indicated that chicken steroidogenic cells are derived from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and downregulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We have identified TOX3 as a previously unreported transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1-positive Leydig cells. TOX3 overexpression in male and female gonads resulted in a significant decline in CYP17A1-positive steroidogenic cells. In ovo knockdown of the testis determinant DMRT1 in male gonads resulted in a downregulation of TOX3 expression. Conversely, DMRT1 overexpression caused an increase in TOX3 expression. Taken together, these data indicate that DMRT1-mediated regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly, via signaling from the supporting to steroidogenic cell populations.

Cell lineage specification and signalling pathway use during development of the lateral plate mesoderm and forelimb mesenchyme.

The lateral plate mesoderm (LPM) is a transient tissue that produces a diverse range of differentiated structures, including the limbs. However, the molecular mechanisms that drive early LPM specification and development are poorly understood. In this study, we use single-cell transcriptomics to define the cell-fate decisions directing LPM specification, subdivision and early initiation of the forelimb mesenchyme in chicken embryos. We establish a transcriptional atlas and global cell-cell signalling interactions in progenitor, transitional and mature cell types throughout the developing forelimb field. During LPM subdivision, somatic and splanchnic LPM fate is achieved through activation of lineage-specific gene modules. During the earliest stages of limb initiation, we identify activation of TWIST1 in the somatic LPM as a putative driver of limb bud epithelial-to-mesenchymal transition. Furthermore, we define a new role for BMP signalling during early limb development, revealing that it is necessary for inducing a somatic LPM fate and initiation of limb outgrowth, potentially through activation of TBX5. Together, these findings provide new insights into the mechanisms underlying LPM development, somatic LPM fate choice and early initiation of the vertebrate limb.

Resolving the mechanisms underlying epithelial-to-mesenchymal transition of the lateral plate mesoderm.

Formation of the vertebrate limb buds begins with a localized epithelial-to-mesenchymal transition (EMT) of the somatic lateral plate mesoderm (LPM). While the processes that drive proliferation and outgrowth of the limb mesenchyme are well established, the fundamental mechanisms that precede this process and initiate EMT are less understood. In this review, we outline putative drivers of EMT of the LPM, drawing from analyses across a range of vertebrates and developmental models. We detail the expression patterns of key EMT transcriptional regulators in the somatic LPM of the presumptive limb fields, and their potential role in producing a mesenchymal cell fate. These include a putative cooperative role between the EMT inducers PRRX1 and TWIST1, supported by evidence in zebrafish and chicken models but unconfirmed data from mice. As such, additional functional data are required to definitively determine the mechanisms that initiate and drive EMT of the somatic LPM, a critical transition preceding formation of the limb bud mesenchyme.

The Curious Case of Avian Sex Determination.

Ioannidis and colleagues show that the gene DMRT1 is the master regulator of testis development in the chicken. Yet, remarkably, when this gene is deleted in genetic males and gonads form ovaries, the body remains male. This debunks the notion that somatic sex is driven primarily by hormones in birds.

An evo-devo perspective of the female reproductive tract.

The vertebrate female reproductive tract has undergone considerable diversification over evolution, having become physiologically adapted to different reproductive strategies. This review considers the female reproductive tract from the perspective of evolutionary developmental biology (evo-devo). Very little is known about how the evolution of this organ system has been driven at the molecular level. In most vertebrates, the female reproductive tract develops from paired embryonic tubes, the Müllerian ducts. We propose that formation of the Müllerian duct is a conserved process that has involved co-option of genes and molecular pathways involved in tubulogenesis in the adjacent mesonephric kidney and Wolffian duct. Downstream of this conservation, genetic regulatory divergence has occurred, generating diversity in duct structure. Plasticity of the Hox gene code and wnt signaling, in particular, may underlie morphological variation of the uterus in mammals, and evolution of the vagina. This developmental plasticity in Hox and Wnt activity may also apply to other vertebrates, generating the morphological diversity of female reproductive tracts evident today.

Characterizing the Residual SCFE Deformity: Utility of the 45-degree Dunn View.

BACKGROUND: After treatment with in situ stabilization, slipped capital femoral epiphysis (SCFE) patients have variable degrees of deformity that can contribute to femoroacetabular impingement (FAI). To evaluate the severity of residual deformity most physicians currently use biplanar radiographs of an anteroposterior pelvis which profiles the lateral head-neck deformity and a frog lateral view which profiles the anterior head-neck deformity. However, the assessment of FAI morphology commonly relies on the 45-degree Dunn view to profile the anterolateral head-neck junction where FAI deformity is maximal. Therefore, the purpose of this study was to compare the magnitude of residual SCFE deformity detected on the frog lateral radiograph to the 45-degree Dunn radiograph. METHODS: A retrospective review of radiographic images of 50 hips (47 patients) diagnosed with SCFE at a single academic institution from 2014 to 2018 was performed. The group included 25 hips evaluated postoperatively after fixation (Group 1) and 25 hips presenting with residual symptomatic SCFE deformity after previous pinning (Group 2). The alpha angle was assessed on both the 45-degree Dunn and frog lateral views for comparison. The modified Southwick slip angle was assessed on the frog lateral view only as a measure of SCFE severity. RESULTS: The Dunn view showed the maximal residual SCFE deformity as measured by the alpha angle in 88% (44/50) of cases. Overall, the mean alpha angle (70.7 degree vs. 60.1 degree, P <0.001) was significantly larger on the Dunn view as compared with frog lateral. Subgroup analysis showed a larger alpha angle on Dunn view compared with frog lateral in both immediate postoperative and residual SCFE deformities as well: Group 1 (69.5 degree vs. 60.9 degree, P <0.001) and Group 2 (71.9 degree vs. 59.3 degree, P <0.001). CONCLUSION: In patients with SCFE, the maximal residual deformity of the proximal femur can typically be seen on the 45-degree Dunn view, rather than on the frog lateral view. Our findings suggest the 45-degree Dunn view may be beneficial part of the postoperative assessment of SCFEs to quantify the true maximal deformity present. LEVEL OF EVIDENCE: Level IV.

Regulation of vertebrate forelimb development and wing reduction in the flightless emu.

The vertebrate limb is a dynamic structure which has evolved into many diverse forms to facilitate complex behavioral adaptations. The principle molecular and cellular processes that underlie development of the vertebrate limb are well characterized. However, how these processes are altered to drive differential limb development between vertebrates is less well understood. Several vertebrate models are being utilized to determine the developmental basis of differential limb morphogenesis, though these typically focus on later patterning of the established limb bud and may not represent the complete developmental trajectory. Particularly, heterochronic limb development can occur prior to limb outgrowth and patterning but receives little attention. This review summarizes the genetic regulation of vertebrate forelimb diversity, with particular focus on wing reduction in the flightless emu as a model for examining limb heterochrony. These studies highlight that wing reduction is complex, with heterochronic cellular and genetic events influencing the major stages of limb development. Together, these studies provide a broader picture of how different limb morphologies may be established during development.

Appraisal and coping predict health and well-being during the COVID-19 pandemic: An international approach.

COVID-19 has had a devastating impact on people worldwide. We conducted an international survey (n = 3646) examining the degree to which people's appraisals and coping activities around the pandemic predicted their health and well-being. We obtained subsamples from 12 countries-Bangladesh, Bulgaria, China, Colombia, India, Israel, the Netherlands, Norway, Peru, Portugal, Turkey and the United States. For each, we assessed appraisals and coping strategies as well as indicators of physical and mental health and well-being. Results indicated that, despite mean-level societal differences in outcomes, the pattern of appraisals and coping strategies predicting health and well-being was consistent across countries. Use of disengagement coping (particularly behavioural disengagement and self-isolation) was associated with relatively negative outcomes. In contrast, optimistic appraisals (particularly of high accommodation-focused coping potential and the ability to meet one's physical needs), use of problem-focused coping strategies (especially problem-solving) and accommodative coping strategies (especially positive reappraisal and self-encouragement) were associated with relatively positive outcomes. Our study highlights the critical importance of considering accommodative coping in stress and coping research. It also provides important information on how people have been dealing with the pandemic, the predictors of well-being under pandemic conditions and the generality of such relations.

Transcriptional landscape of the embryonic chicken Müllerian duct.

BACKGROUND: Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract in vertebrates. Many disorders of female reproduction can be attributed to anomalies of Müllerian duct development. However, the molecular genetics of Müllerian duct formation is poorly understood and most disorders of duct development have unknown etiology. In this study, we describe for the first time the transcriptional landscape of the embryonic Müllerian duct, using the chicken embryo as a model system. RNA sequencing was conducted at 1 day intervals during duct formation to identify developmentally-regulated genes, validated by in situ hybridization. RESULTS: This analysis detected hundreds of genes specifically up-regulated during duct morphogenesis. Gene ontology and pathway analysis revealed enrichment for developmental pathways associated with cell adhesion, cell migration and proliferation, ERK and WNT signaling, and, interestingly, axonal guidance. The latter included factors linked to neuronal cell migration or axonal outgrowth, such as Ephrin B2, netrin receptor, SLIT1 and class A semaphorins. A number of transcriptional modules were identified that centred around key hub genes specifying matrix-associated signaling factors; SPOCK1, HTRA3 and ADGRD1. Several novel regulators of the WNT and TFG-ß signaling pathway were identified in Müllerian ducts, including APCDD1 and DKK1, BMP3 and TGFBI. A number of novel transcription factors were also identified, including OSR1, FOXE1, PRICKLE1, TSHZ3 and SMARCA2. In addition, over 100 long non-coding RNAs (lncRNAs) were expressed during duct formation. CONCLUSIONS: This study provides a rich resource of new candidate genes for Müllerian duct development and its disorders. It also sheds light on the molecular pathways engaged during tubulogenesis, a fundamental process in embryonic development.

Validation of the Health-Related Felt Stigma and Concealment Questionnaire.

OBJECTIVE: Stigma is associated with many health conditions, including chronic pain. Research on health-related stigma is limited by the lack of validated instruments that distinguish among various stigma-related constructs. We aimed to develop and validate such a measure for pediatric functional abdominal pain (FAP). Felt stigma (FS) was defined as comprising both perceived and internalized stigma. Stigma concealment (SC) was defined as efforts by stigmatized individuals to prevent others from learning of their condition. METHODS: Using a theory-driven approach, we adapted items from existing self-report measures of stigma to construct the health-related FS and Concealment Questionnaire (FSC-Q). Patients with FAP (N = 179, ages 11-17) completed the preliminary FSC-Q and health-related measures hypothesized to be associated with stigma. Cognitive interviewing and exploratory factor analysis (EFA) informed the final version of the measure. RESULTS: EFA identified a 2-factor model comprised of FS and SC. The FS and SC scales exhibited good internal consistency and construct validity. Consistent with study hypotheses, both factors were significantly associated with anxiety, depression, pain catastrophizing, pain threat, physical symptoms, and pain interference/disability. Higher FS was associated with higher mental healthcare utilization. The subset of participants meeting criteria for irritable bowel syndrome (IBS) reported higher FS and SC compared with those without IBS. CONCLUSION: The FSC-Q may help advance research on health-related stigma in FAP and other chronic health conditions by allowing for assessment of distinct stigma-related constructs.

Profiles of appraisal, motivation, and coping for positive emotions.

We used a retrospective survey (N = 346) to model the patterns of appraisal, motivation, and coping that uniquely correspond with 12 positive emotions (affection/love, amusement, awe, challenge/determination, compassion, gratitude, happiness/joy, hope, interest, pride, relief, and serenity/tranquillity). Generally, we conceptually replicated previously demonstrated appraisal profiles of positive emotion while also examining how additional appraisals differentiate among positive emotions. We then uncovered the motivational goals and coping processes associated with each positive emotion. We discuss the implications of our findings for future research on positive emotion.

Applying Single-Cell Analysis to Gonadogenesis and DSDs (Disorders/Differences of Sex Development).

The gonads are unique among the body's organs in having a developmental choice: testis or ovary formation. Gonadal sex differentiation involves common progenitor cells that form either Sertoli and Leydig cells in the testis or granulosa and thecal cells in the ovary. Single-cell analysis is now shedding new light on how these cell lineages are specified and how they interact with the germline. Such studies are also providing new information on gonadal maturation, ageing and the somatic-germ cell niche. Furthermore, they have the potential to improve our understanding and diagnosis of Disorders/Differences of Sex Development (DSDs). DSDs occur when chromosomal, gonadal or anatomical sex are atypical. Despite major advances in recent years, most cases of DSD still cannot be explained at the molecular level. This presents a major pediatric concern. The emergence of single-cell genomics and transcriptomics now presents a novel avenue for DSD analysis, for both diagnosis and for understanding the molecular genetic etiology. Such -omics datasets have the potential to enhance our understanding of the cellular origins and pathogenesis of DSDs, as well as infertility and gonadal diseases such as cancer.

Association of Functional Screening Tests and Noncontact Injuries in Division I Women Student-Athletes.

Warren, M, Lininger, M, Smith, CA, Copp, A, and Chimera, NJ. Association of functional screening tests and noncontact injuries in Division I women student-athletes. J Strength Cond Res 34(8): 2302-2311, 2020-To determine the association between functional screening tests and lower-body, noncontact injuries in Division I women basketball, soccer, and volleyball student-athletes (SA). Sixty-eight injury-free women SA (age: 19.1 ± 1.1 years, height: 171.3 ± 8.7 cm, and mass: 68.4 ± 9.5 kg) were tested preseason with single hop (SH), triple hop (TH), and crossover hop (XH) for distance, and isometric hip strength (abduction, extension, and external rotation) in randomized order. The first lower-body (spine and lower extremity), noncontact injury requiring intervention by the athletic trainer was abstracted from the electronic medical record. Receiver operating characteristic and area under the curve (AUC) were calculated to determine cut-points for each hopping test from the absolute value of between-limb difference. Body mass-adjusted strength was categorized into tertiles. Logistic regression determined the odds of injury with each functional screening test using the hopping tests cut-points and strength categories, adjusting for previous injury. Fifty-two SA were injured during the sport season. The cut-point for SH was 4 cm (sensitivity = 0.77, specificity = 0.43, and AUC = 0.53), and for TH and XH was 12 cm (sensitivity = 0.75 and 0.67, specificity = 0.71 and 0.57, AUC = 0.59 and 0.41, respectively). A statistically significant association with TH and injuries (adjusted odds ratio = 6.50 [95% confidence interval: 1.69-25.04]) was found. No significant overall association was found with SH or XH, nor with the strength tests. Using a clinically relevant injury definition, the TH showed the strongest predictive ability for noncontact injuries. This hopping test may be a clinically useful tool to help identify increased risk of injury in women SA participating in high-risk sports.

Is Resolution of Chronic Pain Associated With Changes in Blood Pressure-related Hypoalgesia?

Background: In healthy individuals, elevated resting blood pressure (BP) is associated with reduced pain responsiveness and lower temporal summation. Prior work indicates that this BP-related hypoalgesia is reduced in individuals with chronic pain. Purpose: This study evaluated whether resolution of chronic pain was associated with greater BP-related hypoalgesia compared to nonresolution. Methods: From a prospective sample of adolescents and young adults diagnosed with chronic functional abdominal pain an average of 9 years earlier, 99 individuals in whom the condition had resolved and 51 individuals with ongoing abdominal pain were studied. Resting systolic BP was assessed, followed by evaluation of thermal pain threshold and tolerance, and assessment of temporal summation to thermal pain stimuli. Results: Higher resting systolic BP was significantly associated with higher pain threshold and tolerance, and lower temporal summation only in the group with resolved functional abdominal pain (p < .05). Hierarchical regressions revealed that interactions between BP and resolution of chronic pain were significant only for pain tolerance (p < .05). Analyses by sex indicated that interactions between BP and resolution status were significant for the temporal summation outcome in males but not in females. Conclusions: Results suggest that BP-related hypoalgesic mechanisms may be more effective in individuals in whom chronic pain has resolved compared to those with ongoing chronic pain. Findings hint at sex differences in the extent to which resolution of chronic pain is associated with BP-related hypoalgesia. Whether greater BP-related hypoalgesia is a consequence of, or alternatively a contributor to, resolution of chronic pain warrants further investigation.

Intrawound Vancomycin Powder Reduces Early Prosthetic Joint Infections in Revision Hip and Knee Arthroplasty.

Intrawound vancomycin powder has shown efficacy and safety in decreasing postoperative spine infections, but its use in arthroplasty has not been well established. The purpose of this study was to compare the rate of early prosthetic joint infections (PJI) with and without the use of intrawound vancomycin powder during joint arthroplasty. A retrospective cohort of all patients who underwent primary or revision hip or knee arthroplasty by two surgeons over a two-year period at a single hospital system was evaluated. The control group received standard systemic prophylaxis only, whereas the treatment group received 1 g of vancomycin powder in the surgical wound in addition to systemic prophylaxis. A statistically significant decrease in the overall PJI rate was found in the treatment group (4/816=0.49%) compared to the control group (13/824=1.57%; p=0.0479). Subgroup analysis demonstrated a trend toward fewer PJIs in the vancomycin group, however, only the revision procedures showed a statistically significant reduction in early PJIs after the initiation of vancomycin (7/180=3.89% to 0/134=0%; p=0.0217). The use of intrawound vancomycin powder was associated with a significant reduction in the overall incidence of early PJIs following joint arthroplasty, however, only the revision procedures demonstrated a significant reduction in the rate of early PJIs.

Tuck Jump Assessment: An Exploratory Factor Analysis in a College Age Population.

Lininger, MR, Smith, CA, Chimera, NJ, Hoog, P, and Warren, M. Tuck Jump Assessment: An exploratory factor analysis in a college age population. J Strength Cond Res 31(3): 653-659, 2017-Due to the high rate of noncontact lower extremity injuries that occur in the collegiate setting, medical personnel are implementing screening mechanisms to identify those athletes that may be at risk for certain injuries before starting a sports season. The tuck jump assessment (TJA) was created as a "clinician friendly" tool to identify lower extremity landing technique flaws during a plyometric activity. There are 10 technique flaws that are assessed as either having the apparent deficit or not during the TJA. Technique flaws are then summed up for an overall score. Through expert consensus, these 10 technique flaws have been grouped into 5 modifiable risk factors: ligament dominance, quadriceps dominance, leg dominance or residual injury deficits, trunk dominance ("core" dysfunction), and technique perfection. Research has not investigated the psychometric properties of the TJA technique flaws or the modifiable risk factors. The present study is a psychometric analysis of the TJA technique flaws to measure the internal structure using an exploratory factor analysis (EFA) using data from collegiate athletes (n = 90) and a general college cohort (n = 99). The EFA suggested a 3 factor model accounting for 46% of the variance. The 3 factors were defined as fatigue, distal landing pattern, and proximal control. The results differ from the 5 modifiable risk categories as previously suggested. These results may question the use of a single score, a unidimensional construct, of the TJA for injury screening.

Comparison of Female Collegiate Athletes and College Age Cohort in Tuck Jump Assessment.

Smith, CA, Olson, BK, Olson, LA, Chimera, NJ, and Warren, M. Comparison of female collegiate athletes and college age cohort in tuck jump assessment. J Strength Cond Res 31(4): 1048-1054, 2017-The tuck jump assessment (TJA) is a plyometric jumping assessment with 10 flaw criteria against which technique is assessed over a 10-second interval. The TJA has been reported as a tool for identifying neuromuscular deficits that increase risk for anterior cruciate ligament injury, but group specific data on female TJA scores are limited. No cut point has been developed for groups with different activity levels or participation in athletics. This study investigated the association between TJA score and athletic participation in college-aged females. One hundred twenty-one females (53 collegiate athletes and 68 college students) completed the TJA. TJA score was the sum of flaws for the 10 criteria observed, and the number of jumps was recorded. Poisson regression was used to assess the association between TJA score and number of jumps. The association between each of the 10 flaws between groups was assessed with the chi-square test. No significant association was found between groups for TJA score (mean ± SD: 4.66 ± 1.07 athletes; 5.45 ± 1.05 college cohort; p = 0.06; ß = 0.82). Athletes jumped significantly more times (12.23 ± 1.04 athletes; 9.35 ± 1.04 college cohort). Athletes had a lower proportion of 2 flaws: "thighs do not reach parallel" and "pause between jumps." Lower statistical power may limit interpretation of the remaining flaws. The lack of control of the number of jumps may impact TJA score. To improve the TJA usefulness on the field and clinic, the protocol may need to standardize the number of jumps.

Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads.

DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 in located on the Z sex chromosome and is currently the best candidate master regulator of avian gonadal sex differentiation. We previously showed that knockdown of DMRT1 expression during the period of sexual differentiation induces feminisation of male embryonic chicken gonads. This gene is therefore necessary for proper testis development in the chicken. However, whether it is sufficient to induce testicular differentiation has remained unresolved. We show here that over-expression of DMRT1 induces male pathway genes and antagonises the female pathway in embryonic chicken gonads. Ectopic DMRT1 expression in female gonads induces localised SOX9 and AMH expression. It also induces expression of the recently identified Z-linked male factor, Hemogen (HEMGN). Masculinised gonads show evidence of cord-like structures and retarded female-type cortical development. Furthermore, expression of the critical feminising enzyme, aromatase, is reduced in the presence of over-expressed DMRT1. These data indicate that DMRT1 is an essential sex-linked regulator of gonadal differentiation in avians, and that it likely acts via a dosage mechanism established through the lack of global Z dosage compensation in birds.

Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

BACKGROUND: Despite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29). RESULTS: RNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue. CONCLUSIONS: This study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.

Anti-Müllerian Hormone Is Required for Chicken Embryonic Urogenital System Growth but Not Sexual Differentiation.

In mammals, the primary role of anti-Müllerian hormone (AMH) during development is the regression of Müllerian ducts in males. These structures otherwise develop into fallopian tubes, oviducts, and upper vagina, as in females. This highly conserved function is retained in birds and is supported by the high levels of AMH expression in developing testes. In mammals, AMH expression is controlled partly by the transcription factor, SOX9. However, in the chicken, AMH mRNA expression precedes that of SOX9 , leading to the view that AMH may lie upstream of SOX9 and play a more central role in avian testicular development. To help define the role of AMH in chicken gonad development, we suppressed AMH expression in chicken embryos using RNA interference. In males, AMH knockdown did not affect the expression of key testis pathway genes, and testis cords developed normally. However, a reduction in the size of the mesonephros and gonads was observed, a phenotype that was evident in both sexes. This growth defect occurred as a result of the reduced proliferative capacity of the cells of these tissues, and male gonads also had a significant reduction in germ cell numbers. These data suggest that although AMH does not directly contribute to testicular or ovarian differentiation, it is required in a sex-independent manner for proper cell proliferation and urogenital system growth.