Mini review: Asymmetric Müllerian duct development in the chicken embryo.

Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract. In humans, the Müllerian ducts differentiate into the Fallopian tubes, uterus and upper portion of the vagina. In birds and reptiles, the Müllerian ducts develop into homologous structures, the oviducts. The genetic and hormonal regulation of duct development is a model for understanding sexual differentiation. In males, the ducts typically undergo regression during embryonic life, under the influence of testis-derived Anti-Müllerian Hormone, AMH. In females, a lack of AMH during embryogenesis allows the ducts to differentiate into the female reproductive tract. In the chicken embryo, a long-standing model for development and sexual differentiation, Müllerian duct development in females in asymmetric. Only the left duct forms an oviduct, coincident with ovary formation only on the left side of the body. The right duct, together with the right gonad, becomes vestigial. The mechanism of this avian asymmetry has never been fully resolved, but is thought to involve local interplay between AMH and sex steroid hormones. This mini-review re-visits the topic, highlighting questions in the field and proposing a testable model for asymmetric duct development. We argue that current molecular and imaging techniques will shed new light on this curious asymmetry. Information on asymmetric duct development in the chicken model will inform our understanding of sexual differentiation in vertebrates more broadly.

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.

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.

Twenty-year Outcomes of a Pediatric Chronic Abdominal Pain Cohort: Early Adulthood Health Status and Offspring Physical and Behavioral Health.

Chronic abdominal pain (CAP) represents a common pediatric primary pain disorder that can have long-term effects on physical and mental health into adulthood. Pediatric CAP and Control cohorts recruited in childhood (∼11 years old, T1) and then assessed in emerging adulthood (∼20 years old, T2) were evaluated again for health outcomes in early adulthood (∼30 years old, T3) for the current study. Further, the study evaluated the mental and physical health of offspring of participants who had become parents. Participants who agreed to enroll at T3 (CAP: n = 90, Control: n = 55) completed measures regarding current health, health-related quality of life (HRQoL), and their child's health when applicable. Results indicated close to 20% of the CAP cohort reported recurrent CAP across all 3 timepoints. Participants with current CAP reported poorer HRQoL compared to participants with remitted CAP who reported poorer HRQoL compared to Control participants. The CAP cohort reported higher health-related anxiety compared to the Control cohort regardless of current pain status. CAP compared to Control participants reported greater emotional problems and fewer conduct problems in their children. Longitudinal studies are needed to assess the developmental course of pediatric chronic pain and intergenerational pathways of risk and resilience. Perspective: This article evaluates patterns of chronic abdominal pain from childhood into early adulthood. Patients with pediatric chronic abdominal pain continue to present with health-related anxiety in adulthood and report greater emotional problems in offspring.

Fadrozole-mediated sex reversal in the embryonic chicken gonad involves a PAX2 positive undifferentiated supporting cell state.

Gonadal sex differentiation among vertebrates involves divergent fates of a common group of progenitor cells present in both presumptive ovaries and testes. The first cell type to differentiate gives rise to pre-Sertoli cells in the testis, and pre-follicular cells in the ovary. These cells derive from a common lineage of so-called "supporting cells". In birds and other egg-laying vertebrates, locally synthesised estrogen has a central role in ovarian development and influences the fate of these supporting cells. Manipulation of estrogen levels during embryonic development induces gonadal sex reversal, providing an experimental setting to evaluate the process of gonadal sex differentiation. Recently, we identified PAX2 as a novel marker of the undifferentiated supporting cell lineage in the chicken embryo, expressed in both sexes prior to overt gonadal sex differentiation. PAX2 expression is downregulated at the onset of gonadal sex differentiation in both males and females. The analysis of this undifferentiated supporting cell marker, together with Sertoli (male) and pre-granulosa (female) will enhance our understanding of supporting cell differentiation. Here we characterized the supporting cells differentiation process and identified undifferentiated supporting cells in estrogen-mediated sex reversal experiments. Female embryos treated with the aromatase inhibitor fadrozole developed into ovotestis, containing pre-granulosa cells, Sertoli cells and PAX2 positive undifferentiated supporting cells. In contrast, male embryos treated with 17ß-estradiol showed no PAX2+ undifferentiated gonadal supporting cells. Fadrozole time-course as well as multiple dose analysis suggests that supporting cell transdifferentiation involves a dedifferentiation event into a PAX2+ undifferentiated supporting cell state, followed by a redifferentiation towards the opposite sex lineage.

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.

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.

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.

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.

Internet-delivered cognitive behavioral therapy for youth with functional abdominal pain: a randomized clinical trial testing differential efficacy by patient subgroup.

ABSTRACT: Inconsistent results of psychological treatments for pediatric functional abdominal pain (FAP) may be due to heterogeneity of patients' pain-related psychological characteristics. This randomized controlled trial tested whether statistically derived patient subgroups (high pain dysfunctional [HPD], high pain adaptive [HPA], and low pain adaptive [LPA]) moderated response to cognitive behavior therapy (CBT) for adolescents with FAP and their parents (n = 278 dyads; patients were 66% female, mean [SD] age was 14.62 [1.88] years, and parents were 95% female). Randomization to Internet-delivered CBT vs Internet-delivered pain education (EDU) was stratified by patient subgroup. Follow-up assessments of gastrointestinal (GI) symptoms (primary outcome), abdominal pain, and pain interference were at midtreatment, posttreatment, 6 months, and 12 months. Data were analysed using linear mixed effects models. Significant treatment × subgroup × time interaction effects showed that patient subgroup significantly moderated the effect of treatment on GI symptoms (t[853 = -2.93, P = 0.003) and abdominal pain (t(844) = -2.14, P = 0.03) across the treatment period. Among HPD youth, those in CBT had significantly greater GI symptom reduction than those in EDU through posttreatment. By contrast, among HPA and LPA youth, symptom improvement did not differ by treatment condition. Furthermore, among all patients assigned to CBT, HPD youth demonstrated significantly greater reductions in GI symptoms compared with HPA and LPA youth and greater reductions in abdominal pain compared with LPA youth. All subgroups maintained symptom reductions throughout the follow-up period. Results suggest that subgrouping FAP patients may inform treatment allocation and optimize treatment response.

PAX2 + Mesenchymal Origin of Gonadal Supporting Cells Is Conserved in Birds.

During embryonic gonadal development, the supporting cell lineage is the first cell type to differentiate, giving rise to Sertoli cells in the testis and pre-granulosa cells in the ovary. These cells are thought to direct other gonadal cell lineages down the testis or ovarian pathways, including the germline. Recent research has shown that, in contrast to mouse, chicken gonadal supporting cells derive from a PAX2/OSR1/DMRT1/WNT4 positive mesenchymal cell population. These cells colonize the undifferentiated genital ridge during early gonadogenesis, around the time that germ cells migrate into the gonad. During the process of somatic gonadal sex differentiation, PAX2 expression is down-regulated in embryonic chicken gonads just prior to up-regulation of testis- and ovary-specific markers and prior to germ cell differentiation. Most research on avian gonadal development has focused on the chicken model, and related species from the Galloanserae clade. There is a lack of knowledge on gonadal sex differentiation in other avian lineages. Comparative analysis in birds is required to fully understand the mechanisms of avian sex determination and gonadal differentiation. Here we report the first comparative molecular characterization of gonadal supporting cell differentiation in birds from each of the three main clades, Galloanserae (chicken and quail), Neoaves (zebra finch) and Palaeognathe (emu). Our analysis reveals conservation of PAX2+ expression and a mesenchymal origin of supporting cells in each clade. Moreover, down-regulation of PAX2 expression precisely defines the onset of gonadal sex differentiation in each species. Altogether, these results indicate that gonadal morphogenesis is conserved among the major bird clades.

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.

Anatomy, Endocrine Regulation, and Embryonic Development of the Rete Testis.

Reproduction in males requires the transfer of spermatozoa from testis tubules via the rete system to the efferent ductules, epididymis, and vas deferens. The rete therefore forms an essential bridging system between the testis and excurrent ducts. Yet the embryonic origin and molecular regulation of rete testis development is poorly understood. This review examines the anatomy, endocrine control, and development of the mammalian rete testis, focusing on recent findings on its molecular regulation, identifying gaps in our knowledge, and identifying areas for future research. The rete testis develops in close association with Sertoli cells of the seminiferous cords, although unique molecular markers are sparce. Most recently, modern molecular approaches such as global RNA-seq have revealed the transcriptional signature of rete cell precursors, pointing to at least a partial common origin with Sertoli cells. In the mouse, genes involved in Sertoli cell development or maintenance, such as Sox9, Wt1, Sf1, and Dmrt1, are also expressed in cells of the rete system. Rete progenitor cells also express unique markers, such as Pax8, E-cadherin, and keratin 8. These must directly or indirectly regulate the physical joining of testis tubules to the efferent duct system and confer other physiological functions of the rete. The application of technologies such as single-cell RNA-seq will clarify the origin and developmental trajectory of this essential component of the male reproductive tract.

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.

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.

Subgroups of Pediatric Patients With Functional Abdominal Pain: Replication, Parental Characteristics, and Health Service Use.

OBJECTIVES: Prior work in a cohort of youth with functional abdominal pain (FAP) identified patient subgroups (High Pain Dysfunctional, High Pain Adaptive, Low Pain Adaptive) that predicted differences in the course of FAP from childhood into young adulthood. We aimed to replicate these subgroups in a new sample of adolescents with FAP using the original classification algorithm and to extend subgroup characteristics to include parental characteristics and health service use. METHODS: Adolescents (n=278; ages 11 to 17 y, 66% females) presenting to a gastroenterology clinic for abdominal pain, and their parents (92% mothers) completed self-report measures; adolescents also completed a 7-day pain diary. RESULTS: The replicated patient subgroups exhibited distress and impairment similar to subgroups in the original sample. Moreover, in novel findings, the High Pain Dysfunctional subgroup differed from other subgroups by the predominance of mother-daughter dyads jointly characterized by high levels of anxiety, depressive symptoms, pain behavior, and pain catastrophizing. The High Pain Dysfunctional subgroup used more health care services than Low Pain Adaptive but did not differ from High Pain Adaptive. DISCUSSION: Findings replicate and extend the original FAP classification and suggest that the subgroups have unique patient and parent features that may reflect distinct illness mechanisms requiring different treatments.

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.

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.