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.

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.

Treatment patterns and decision drivers to discharge patients with depression hospitalised for acute suicidal ideation in Europe.

BACKGROUND: There is limited published information about the management of patients with major depressive disorder (MDD) hospitalised for acute suicidal ideation (SI). This study aimed to identify treatment patterns and unmet needs in the management of these patients and the decision drivers for hospital discharge. METHODS: Cross-sectional survey-based study enrolling hospital-based European psychiatrists. The study had a qualitative and a quantitative stage, including a conjoint exercise. RESULTS: Each respondent (N = 413) managed, on average, 62 MDD patients with acute SI per typical three-month period; 76% of these patients required hospitalisation. Severity of SI and severity of MDD were considered the most important factors for hospital admission and discharge. In the conjoint analysis, these attributes accounted for 54% of the discharge decision. Key treatment goals included improving depressive symptoms and achieving MDD remission. Antidepressants were a standard treatment for 98% of respondents but 63% defined rapid onset of action as a critical unmet need, followed by a good tolerability profile (34%). LIMITATIONS: The study has a cross-sectional design representing respondents' behaviour and attitudes at a particular point in time. In the conjoint analysis, the results represent stated behaviour and not observed clinical behaviour. CONCLUSIONS: Physicians' decisions to admit and discharge patients with MDD hospitalised for acute SI are mostly driven by the severity of SI and depression. Antidepressants with rapid onset of action, which can quickly improve depressive symptoms, represent a key unmet need for these patients and may contribute to a higher likelihood of early discharge.

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.

Genetic Regulation of Avian Testis Development.

As in other vertebrates, avian testes are the site of spermatogenesis and androgen production. The paired testes of birds differentiate during embryogenesis, first marked by the development of pre-Sertoli cells in the gonadal primordium and their condensation into seminiferous cords. Germ cells become enclosed in these cords and enter mitotic arrest, while steroidogenic Leydig cells subsequently differentiate around the cords. This review describes our current understanding of avian testis development at the cell biology and genetic levels. Most of this knowledge has come from studies on the chicken embryo, though other species are increasingly being examined. In chicken, testis development is governed by the Z-chromosome-linked DMRT1 gene, which directly or indirectly activates the male factors, HEMGN, SOX9 and AMH. Recent single cell RNA-seq has defined cell lineage specification during chicken testis development, while comparative studies point to deep conservation of avian testis formation. Lastly, we identify areas of future research on the genetics of avian testis development.

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.

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.

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.

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.

Gonadal Sex Differentiation: Supporting Versus Steroidogenic Cell Lineage Specification in Mammals and Birds.

The gonads of vertebrate embryos are unique among organs because they have a developmental choice; ovary or testis formation. Given the importance of proper gonad formation for sexual development and reproduction, considerable research has been conducted over the years to elucidate the genetic and cellular mechanisms of gonad formation and sexual differentiation. While the molecular trigger for gonadal sex differentiation into ovary of testis can vary among vertebrates, from egg temperature to sex-chromosome linked master genes, the downstream molecular pathways are largely conserved. The cell biology of gonadal formation and differentiation has long thought to also be conserved. However, recent discoveries point to divergent mechanisms of gonad formation, at least among birds and mammals. In this mini-review, we provide an overview of cell lineage allocation during gonadal sex differentiation in the mouse model, focusing on the key supporting and steroidogenic cells and drawing on recent insights provided by single cell RNA-sequencing. We compare this data with emerging information in the chicken model. We highlight surprising differences in cell lineage specification between species and identify gaps in our current understanding of the cell biology underlying gonadogenesis.

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.

FOXL2 antagonises the male developmental pathway in embryonic chicken gonads.

FOXL2 is a conserved transcription factor with a central role in ovarian development and function. Studies in humans and mice indicate that the main role of FOXL2 is in the postnatal ovary, namely folliculogenesis. To shed light on the function and evolution of FOXL2 in the female gonad, we examined its role in embryonic avian gonads, using in ovo over-expression and knockdown. FOXL2 mRNA and protein are expressed female-specifically in the embryonic chicken gonad, just prior to the onset of sexual differentiation. FOXL2 is expressed in the medullary cord cells, in the same cell type as aromatase (CYP19A1). In addition, later in development, expression also becomes localised in a subset of cortical cells, distinct from those expressing estrogen receptor alpha. Mis-expression of FOXL2 in the male chicken embryonic gonad suppresses the testis developmental pathway, abolishing local expression of the male pathway genes, SOX9, DMRT1 and AMH, and repressing Sertoli cell development. Conversely, knockdown of FOXL2 expression allows ectopic activation of SOX9 in female gonads. However, mis-expression of FOXL2 alone was insufficient to activate aromatase expression in male gonads, while FOXL2 knockdown did not affect aromatase expression in females. These results indicate that FOXL2 plays an important role in embryonic differentiation of the avian ovary via antagonism of SOX9, but may be dispensable for aromatase activation at embryonic stages. The data suggest that FOXL2 has different roles in different species, more central for embryonic ovarian differentiation in egg-laying vertebrates.

Dynamic paraspeckle component localisation during spermatogenesis.

Expression profiles and subcellular localisations of core Drosophila behaviour/human splicing (DBHS) proteins (PSPC1, SFPQ and NONO) and NEAT1, a long noncoding RNA (lncRNA), are investigated in developing and adult mouse testes. Core DBHS proteins are markers for the distinct subnuclear domain termed paraspeckles, while a long NEAT1 isoform scaffold facilitates paraspeckle nucleation. Paraspeckles contain many proteins (>40) and are broadly involved in RNA metabolism, including transcriptional regulation by protein sequestration, nuclear retention of A-to-I edited RNA transcripts to regulate translation and promoting survival during cellular stress. Immunohistochemistry reveals cell-specific profiles for core DBHS paraspeckle protein expression, indicating their functional diversity. PSPC1 is an androgen receptor co-activator, and it is detected in differentiating Sertoli cell nuclei from day 15 onwards, as they develop androgen responsiveness. PSPC1 is nuclear in the most mature male germ cell type present at each age, from foetal to adult life. In adult mouse testes, PSPC1 and SFPQ are present in Sertoli cells, spermatocytes and round spermatids, while the NEAT1 lncRNA appears in the punctate nuclear foci delineating paraspeckles only within Leydig cells. Identification of NEAT1 in the cytoplasm of spermatogonia and spermatocytes must reflect non-paraspeckle-related functions. NONO was absent from germ cells but nuclear in Sertoli cells. Reciprocal nuclear profiles of PSPC1 and γ-H2AX in spermatogenic cells suggest that each performs developmentally regulated roles in stress responses. These findings demonstrate paraspeckles and paraspeckle-related proteins contribute to diverse functions during testis development and spermatogenesis.

A Randomized Controlled Trial using iTClamp, Direct Pressure, and Balloon Catheter Tamponade to Control Neck Hemorrhage in a Perfused Human Cadaver Model.

BACKGROUND: Penetrating neck wounds are common in the civilian and military realms. Whether high or low velocity, they carry a substantial morbidity and mortality rate. OBJECTIVES: We endeavored to ascertain whether the iTClamp is equivalent to direct manual pressure (DMP) and Foley catheter balloon tamponade (BCT). METHODS: Using a perfused cadaver, a 4.5-cm wound was made in Zone 2 of the neck with a 1-cm carotid arteriotomy. Each of the hemorrhage control modalities was randomized and then applied to the wound separately. Time to apply the device and fluid loss with and without neck motion was recorded. RESULTS: There was no significant difference between the fluid loss/no movement (p > 0.450) and fluid loss/movement (p > 0.215) between BCT and iTClamp. There was significantly more fluid lost with DMP than iTClamp with no movement (p > 0.000) and movement (p > 0.000). The iTClamp was also significantly faster to apply than the Foley (p > 0.000). CONCLUSIONS: The iTClamp and BCT were associated with significantly less fluid loss than DMP in a perfused cadaver model. The iTClamp required significantly less time to apply than the BCT. Both the iTClamp and the BCT were more effective than simple DMP. The iTClamp offers an additional option for managing hard-to-control bleeding in the neck.

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.

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.

The cell biology and molecular genetics of Müllerian duct development.

The Müllerian ducts are part of the embryonic urogenital system. They give rise to mature structures that serve a critical function in the transport and development of the oocyte and/or embryo. In most vertebrates, both sexes initially develop Müllerian ducts during embryogenesis, but they regress in males under the influence of testis-derived Anti-Müllerian Hormone (AMH). A number of regulatory factors have been shown to be essential for proper duct development, including Bmp and Wnt signaling molecules, together with homeodomain transcription factors such as PAX2 and LIM1. Later in development, the fate of the ducts diverges between males and females and is regulated by AMH and Wnt signaling molecules (duct regression in males) and Hox genes (duct patterning in females). Most of the genes and molecular pathways known to be involved in Müllerian duct development have been elucidated through animal models, namely, the mouse and chicken. In addition, genetic analysis of humans with reproductive tract disorders has further defined molecular mechanisms of duct formation and differentiation. However, despite our current understanding of Müllerian duct development, some questions remain to be answered at the molecular genetic level. This article is categorized under: Early Embryonic Development > Development to the Basic Body Plan.