Human melanocyte development and melanoma dedifferentiation at single-cell resolution.

In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy.

Estrogens and development of the mouse and human external genitalia.

The Jost hypothesis states that androgens are necessary for normal development of the male external genitalia. In this review, we explore the complementary hypothesis that estrogens can elicit abnormal development of male external genitalia. Herein, we review available data in both humans and mice on the deleterious effects of estrogen on external genitalia development, especially during the "window of susceptibility" to exogenous estrogens. The male and female developing external genitalia in both the human and mouse express ESR1 and ESR2, along with the androgen receptor (AR). Human clinical data suggests that exogenous estrogens can adversely affect normal penile and urethral development, resulting in hypospadias. Experimental mouse data also strongly supports the idea that exogenous estrogens cause penile and urethral defects. Despite key differences, estrogen-induced hypospadias in the mouse displays certain morphogenetic homologies to human hypospadias, including disruption of urethral fusion and preputial abnormalities. Timing of estrogenic exposure, or the "window of susceptibility," is an important consideration when examining malformations of the external genitalia in both humans and mice. In addition to a review of normal human and mouse external genital development, this article aims to review the present data on the role of estrogens in normal and abnormal development of the mouse and human internal and external genitalia. Based on the current literature for both species, we conclude that estrogen-dependent processes may play a role in abnormal genital development.

Anatomy of the mouse penis and internal prepuce.

This paper addresses a confusing issue of preputial anatomy of the mouse. The term "internal prepuce" was used in 2013 to describe a preputial structure integral to the mouse glans penis. Subsequently in 2015 the same term was applied by another group to describe entirely different morphology, generating confusion in the literature. Because it is inappropriate to use the same term to describe entirely different structures, we take this opportunity to provide further descriptive information on the internal prepuce of the mouse employing gross dissection, analysis of serial histologic section sets, three-dimensional reconstruction, scanning electron microscopy and immunohistochemistry. For this purpose, we review and illustrate the relevant literature and provide some additional new data using standard morphological techniques including immunohistochemistry. The mouse internal prepuce is integral to the glans penis and clearly is involved in sexual function in so far as it contains a major erectile body innervated by penile nerves. The development of the mouse internal prepuce is described for the first time and related to the development of the corpus cavernosum glandis.

Chloride channels regulate differentiation and barrier functions of the mammalian airway.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.

Development of the human prepuce and its innervation.

Development of the human prepuce was studied over the course of 9-17 weeks of gestation in 30 specimens. Scanning electron microscopy revealed subtle surface features that were associated with preputial development, namely the appearance of epidermal aggregates that appeared to be associated with formation of the preputial fold. Transverse and sagittal sections revealed that the epidermis of the glans is considerably thicker than that of the penile shaft. We described a novel morphogenetic mechanism of formation of the preputial lamina, namely the splitting of the thick epidermis of the glans into the preputial lamina and the epidermis via the intrusion of mesenchyme containing red blood cells and CD31-positive blood vessels. This process begins at 10-11 weeks of gestation in the proximal aspect of the glans and extends distally. The process is likely to be androgen-dependent and mediated via androgen receptors strategically localized to the morphogenetic process, but signaling through estrogen receptor may play a role. Estrogen receptor alpha (ESR1) has a very limited expression in the developing human glans and prepuce, while estrogen receptor beta (ESR2) is expressed more broadly in the developing preputial lamina, epidermis and urethra. Examination of the ontogeny of innervation of the glans penis and prepuce reveals the presence of the dorsal nerve of the penis as early as 9 weeks of gestation. Nerve fibers enter the glans penis proximally and extend distally over several weeks to eventually reach the distal aspect of the glans and prepuce by 14-16 weeks of gestation.

Androgen and estrogen receptor expression in the developing human penis and clitoris.

To better understand how the human fetal penis and clitoris grows and remodels, we undertook an investigation to define active areas of cellular proliferation and programmed cell death spatially and temporally during development of human fetal external genitalia from the indifferent stage (8 weeks) to 18 weeks of gestation. Fifty normal human fetal penile and clitoral specimens were examined using macroscopic imaging, scanning electron microscopy and immunohistochemical localization for the cellular proliferation and apoptotic markers, Ki67 and Caspase-3. A number of hot spots of cellular proliferation characterized by Ki67 localization are present in the penis and clitoris especially early in development, most notably in the corporal body, glans, remodeling glanular urethra, the urethral plate, the roof of the urethral groove and the fully formed penile urethra. The 12-fold increase in penile length over 10 weeks of growth from 8 to 18 weeks of gestation based on Ki67 labelling appears to be driven by cellular proliferation in the corporal body and glans. Throughout all ages in both the developing penis and clitoris Ki67 labeling was consistently elevated in the ventral epidermis and ventral mesenchyme relative to the dorsal counterparts. This finding is consistent with the intense morphogenetic activity/remodeling in the ventral half of the genital tubercle in both sexes involving formation of the urethral/vestibular plates, canalization of the urethral/vestibular plates and fusion of the urethral folds to form the penile urethra. Areas of reduced or absent Ki67 staining include the urethral fold epithelium that fuses to form the penile tubular urethra. In contrast, the urethral fold mesenchyme is positive for Ki67. Apoptosis was rarely noted in the developing penis and clitoris; the only area of minimal Caspase-3 localization was in the epithelium of the ventral epithelial glanular channel remodeling.

Androgen-independent events in penile development in humans and animals.

The common view on penile development is that it is androgen-dependent, based first and foremost on the fact that the genital tubercle forms a penis in males and a clitoris in females. However, critical examination of the complex processes involved in human penile development reveals that many individual steps in development of the genital tubercle are common to both males and females, and thus can be interpreted as androgen-independent. For certain developmental events this conclusion is bolstered by observations in androgen-insensitive patients and androgen receptor mutant mice. Events in genital tubercle development that are common to human males and females include: formation of (a) the genital tubercle, (b) the urethral plate, (c) the urethral groove, (d) the glans, (e) the prepuce and (f) the corporal body. For humans 6 of 13 individual developmental steps in penile development were interpreted as androgen-independent. For mice 5 of 11 individual developmental steps were found to be androgen-independent, which were verified through analysis of androgen-insensitive mutants. Observations from development of external genitalia of other species (moles and spotted hyena) provide further examples of androgen-independent events in penile development. These observations support the counter-intuitive idea that penile development involves both androgen-independent and androgen-dependent processes.

Clitoral development in the mouse and human.

The goal of this report is (a) to provide the first detailed description of mouse clitoral development, and (b) to compare mouse and human clitoral development. For this purpose, external genitalia of female mice were examined by wholemount microscopy, histology and immunohistochemistry from 14 days of gestation to 10 days postnatal. Human clitoral development was examined by these techniques as well as by scanning electron microscopy and optical projection tomography from 8 to 19 weeks of gestation. The adult mouse clitoris is an internal organ defined by a U-shaped clitoral lamina whose development is associated with the prenatal medial and distal growth of the female preputial swellings along the sides of the genital tubercle to form the circumferential preputial lamina. Regression of the ventral aspect of the preputial lamina leads to formation of the U-shaped clitoral lamina recognized as early as 17 days of gestation. While the adult U-shaped mouse clitoral lamina is closely associated with the vagina, and it appears to be completely non-responsive to estrogen as opposed to the highly estrogen-responsive vaginal epithelium. The prominent perineal appendage in adult females is prepuce, formed via fusion of the embryonic preputial swellings and is not the clitoris. The human clitoris is in many respects a smaller anatomic version of the human penis having all of the external and internal elements except the urethra. The human clitoris (like the human penis) is derived from the genital tubercle with the clitoral glans projecting into the vaginal vestibule. Adult morphology and developmental processes are virtually non-comparable in the mouse and human clitoris.

Hot spots in fetal human penile and clitoral development.

To better understand how the human fetal penis and clitoris grows and remodels, we undertook an investigation to define active areas of cellular proliferation and programmed cell death spatially and temporally during development of human fetal external genitalia from the indifferent stage (8 weeks) to 18 weeks of gestation. Fifty normal human fetal penile and clitoral specimens were examined using macroscopic imaging, scanning electron microscopy and immunohistochemical localization for the cellular proliferation and apoptotic markers, Ki67 and Caspase-3, respectively. A number of hot spots of cellular proliferation characterized by Ki67 localization are present in the penis and clitoris especially early in development, most notably in the corporal body, glans, remodeling glanular urethra, the urethral plate, the roof of the urethral groove and the fully formed penile urethra. The 12-fold increase in penile length over 10 weeks of growth from 8 to 18 weeks of gestation based on Ki67 labelling appears to be driven by cellular proliferation in the corporal body and glans. Throughout all ages in both the developing penis and clitoris Ki67 labeling was consistently elevated in the ventral epidermis and ventral mesenchyme relative to the dorsal counterparts. This finding is consistent with the intense morphogenetic activity/remodeling in the ventral half of the genital tubercle in both sexes involving formation of the urethral/vestibular plates, canalization of the urethral/vestibular plates and fusion of the urethral folds to form the penile urethra. Areas of reduced or absent Ki67 staining include the urethral fold epithelium that fuses to form the penile tubular urethra. In contrast, the urethral fold mesenchyme is positive for Ki67. Apoptosis was rarely noted in the developing penis and clitoris; the only area of minimal Caspase-3 localization was in the epithelium of the ventral epithelial glanular channel remodeling.

Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy.

Technological advances in three-dimensional (3D) reconstruction techniques have previously enabled paradigm shifts in our understanding of human embryonic and fetal development. Light sheet fluorescence microscopy (LSFM) is a recently-developed technique that uses thin planes of light to optically section whole-mount cleared and immunolabeled biologic specimens. The advent of commercially-available light sheet microscopes has facilitated a new generation of research into protein localization and tissue dynamics at extremely high resolution. Our group has applied LSFM to study developing human fetal external genitalia, internal genitalia and kidneys. This review describes LSFM and presents our group's technique for preparing, clearing, immunostaining and imaging human fetal urogenital specimens. We then present light sheet images and videos of each element of the developing human urogenital system. To the extent of our knowledge, the work conducted by our laboratory represents the first description of a method for performing LSFM on the full human urogenital system during the embryonic and fetal periods.

Reproductive tract biology: Of mice and men.

The study of male and female reproductive tract development requires expertise in two separate disciplines, developmental biology and endocrinology. For ease of experimentation and economy, the mouse has been used extensively as a model for human development and pathogenesis, and for the most part similarities in developmental processes and hormone action provide ample justification for the relevance of mouse models for human reproductive tract development. Indeed, there are many examples describing the phenotype of human genetic disorders that have a reasonably comparable phenotype in mice, attesting to the congruence between mouse and human development. However, anatomic, developmental and endocrinologic differences exist between mice and humans that (1) must be appreciated and (2) considered with caution when extrapolating information between all animal models and humans. It is critical that the investigator be aware of both the similarities and differences in organogenesis and hormone action within male and female reproductive tracts so as to focus on those features of mouse models with clear relevance to human development/pathology. This review, written by a team with extensive expertise in the anatomy, developmental biology and endocrinology of both mouse and human urogenital tracts, focusses upon the significant human/mouse differences, and when appropriate voices a cautionary note regarding extrapolation of mouse models for understanding development of human male and female reproductive tracts.

A critical role for estrogen signaling in penis development.

Hypospadias, a developmental defect of the penis, is one of the most common congenital malformations in humans. Its incidence has rapidly increased over recent decades, and this has been largely attributed to our increased exposure to endocrine-disrupting chemicals. Penis development is primarily an androgen-driven process; however, estrogen and xenoestrogens are known to affect penis development in both humans and mice. Here, we investigated the role of estrogen in the developing penis. Using a novel penis culture system, we showed that exogenous estrogen directly targets the developing penis in utero to cause hypospadias. In addition, we also uncovered an unexpected endogenous role for estrogen in normal postnatal penis development and showed that a loss of estrogen signaling results in a mild hypospadias phenotype, the most common manifestation of this disease in humans. Our findings demonstrated that both androgen and estrogen signaling are intrinsically required for normal urethral closure. These findings confirmed that penis development is not an entirely androgen-driven process but one in which endogenous estrogen signaling also plays a critical role.-Govers, L. C., Phillips, T. R., Mattiske, D. M., Rashoo, N., Black, J. R., Sinclair, A., Baskin, L. S., Risbridger, G. P., Pask, A. J. A critical role for estrogen signaling in penis development.

Australian Clinical Consensus Guideline: The diagnosis and acute management of childhood stroke.

Stroke is among the top 10 causes of death in children and survivors carry resulting disabilities for decades, at substantial cost to themselves and their families. Children are not currently able to access reperfusion therapies, due to limited evidence supporting safety and efficacy and long diagnostic delays. The Australian Clinical Consensus Guideline for the Diagnosis and Acute Management of Childhood Stroke was developed to minimize unwarranted variations in care and document best evidence on the risk factors, etiologies, and conditions mimicking stroke that differ from adults. Clinical questions were formulated to inform systematic database searches from 2007 to 2017, limited to English and pediatric studies. SIGN methodology and the National Health and Medical Research Council system were used to screen and classify the evidence. The Grades of Recommendation, Assessment, Development, and Evaluation system (GRADE) was used to grade evidence as strong or weak. The Guideline provides more than 60 evidence-based recommendations to assist prehospital and acute care clinicians in the rapid identification of childhood stroke, choice of initial investigation, to confirm diagnosis, determine etiology, selection of the most appropriate interventions to salvage brain at risk, and prevent recurrence. Recommendations include advice regarding the management of intracranial pressure and congenital heart disease. Implementation of the Guideline will require reorganization of prehospital and emergency care systems, including the development of regional stroke networks, pediatric Code Stroke, rapid magnetic resonance imaging and accreditation of primary pediatric stroke centers with the capacity to offer reperfusion therapies. The Guideline will allow auditing to benchmark timelines of care, access to acute interventions, and outcomes. It will also facilitate the development of an Australian childhood stroke registry, with data linkage to international registries, to allow for accurate data collection on stroke incidence, treatment, and outcomes.

Immunohistochemical expression analysis of the human fetal lower urogenital tract.

We have studied the ontogeny of the developing human male and female urogenital tracts from 9 weeks (indifferent stage) to 16 weeks (advanced sex differentiation) of gestation by immunohistochemistry on mid-sagittal sections. Sixteen human fetal pelvises were serial sectioned in the sagittal plane and stained with antibodies to epithelial, muscle, nerve, proliferation and hormone receptor markers. Key findings are: (1) The corpus cavernosum in males and females extends into the glans penis and clitoris, respectively, during the ambisexual stage (9 weeks) and thus appears to be an androgen-independent event. (2) The entire human male (and female) urethra is endodermal in origin based on the presence of FOXA1, KRT 7, uroplakin, and the absence of KRT10 staining. The endoderm of the urethra interfaces with ectodermal epidermis at the site of the urethral meatus. (3) The surface epithelium of the verumontanum is endodermal in origin (FOXA1-positive) with a possible contribution of Pax2-positive epithelial cells implying additional input from the Wolffian duct epithelium. (4) Prostatic ducts arise from the endodermal (FOXA1-positive) urogenital sinus epithelium near the verumontanum. (5) Immunohistochemical staining of mid-sagittal and para-sagittal sections revealed the external anal sphincter, levator ani, bulbospongiosus muscle and the anatomic relationships between these developing skeletal muscles and organs of the male and female reproductive tracts. Future studies of normal human developmental anatomy will lay the foundation for understanding congenital anomalies of the lower urogenital tract.

Development of the human bladder and ureterovesical junction.

The urinary bladder collects urine from the kidneys and stores it until the appropriate moment for voiding. The trigone and ureterovesical junctions are key to bladder function, by allowing one-way passage of urine into the bladder without obstruction. Embryological development of these structures has been studied in multiple animal models as well as humans. In this report we review the existing literature on bladder development and cellular signalling with particular focus on bladder development in humans. The bladder and ureterovesical junction form primarily during the fourth to eighth weeks of gestation, and arise from the primitive urogenital sinus following subdivision of the cloaca. The bladder develops through mesenchymal-epithelial interactions between the endoderm of the urogenital sinus and mesodermal mesenchyme. Key signalling factors in bladder development include shh, TGF-ß, Bmp4, and Fgfr2. A concentration gradient of shh is particularly important in development of bladder musculature, which is vital to bladder function. The ureterovesical junction forms from the interaction between the Wolffian duct and the bladder. The ureteric bud arises from the Wolffian duct and is incorporated into the developing bladder at the trigone. It was previously thought that the trigonal musculature developed primarily from the Wolffian duct, but it has been shown to develop primarily from bladder mesenchyme. Following emergence of the ureters from the Wolffian ducts, extensive epithelial remodelling brings the ureters to their final trigonal positions via vitamin A-induced apoptosis. Perturbation of this process is implicated in clinical obstruction or urine reflux. Congenital malformations include ureteric duplication and bladder exstrophy.

Three-dimensional imaging of the developing human fetal urogenital-genital tract: Indifferent stage to male and female differentiation.

Recent studies in our lab have utilized three imaging techniques to visualize the developing human fetal urogenital tract in three dimensions: optical projection tomography, scanning electron microscopy and lightsheet fluorescence microscopy. We have applied these technologies to examine changes in morphology and differential gene expression in developing human external genital specimens from the ambisexual stage (<9 weeks fetal age) to well-differentiated male and female organs (>13 weeks fetal age). This work outlines the history and function of each of these three imaging modalities, our methods to prepare specimens for each and the novel findings we have produced thus far. We believe the images in this paper of human fetal urogenital organs produced using lightsheet fluorescence microscopy are the first published to date.

Macroscopic whole-mounts of the developing human fetal urogenital-genital tract: Indifferent stage to male and female differentiation.

We present a detailed review of fetal development of the male and female human urogenital tract from 8 to 22 weeks gestation at the macroscopic and morphometric levels. Human fetal specimens were sexed based on macroscopic identification of fetal testes or ovaries, Wolffian or Müllerian structures and the presence of the SRY gene in the specimens at or near the indifferent stage (8-9 weeks). Specimens were photographed using a dissecting microscope with transmitted and reflected light. Morphometric measurements were taken of each urogenital organ. During this time period, development of the male and female urogenital tracts proceeded from the indifferent stage to differentiated organs. The kidneys, ureters, and bladder developed identically, irrespective of sex with the same physical dimensions and morphologic appearance. The penis, prostate and testis developed in males and the clitoris, uterus and ovary in females. Androgen-dependent growth certainly influenced size and morphology of the penile urethra and prostate, however, androgen-independent growth also accounted for substantial growth in the fetal urogenital tract including the clitoris.

Human glans and preputial development.

The urethra within the human penile shaft develops via (1) an "Opening Zipper" that facilitates distal canalization of the solid urethral plate to form a wide urethral groove and (2) a "Closing Zipper" that facilitates fusion of the epithelial surfaces of the urethral folds. Herein, we extend our knowledge by describing formation of the human urethra within the glans penis as well as development of the prepuce. Forty-eight normal human fetal penile specimens were examined using scanning electron microscopy and optical projection tomography. Serial histologic sections were evaluated for morphology and immunohistochemical localization for epithelial differentiation markers: Cytokeratins 6, 7, 10, FoxA1, uroplakin and the androgen receptor. As the closing zipper completes fusion of the urethral folds within the penile shaft to form a tubular urethra (~ 13 weeks), canalization of the urethral plate continues in proximal to distal fashion into the glans penis to directly form the urethra within the glans without forming an open urethral groove. Initially, the urethral plate is attached ventrally to the epidermis via an epithelial seam, which is remodeled and eliminated, thus establishing mesenchymal confluence ventral to the glanular urethra. The morphogenetic remodeling involves the strategic expression of cytokeratin 7, FoxA1 and uroplakin in endodermal epithelial cells as the tubular glanular urethra forms. The most ventral epithelial cells of the urethral plate are pinched off from the glanular urethra and are reabsorbed into the epidermis ultimately losing expression of their markers, a process undoubtedly regulated by androgens. The prepuce initially forms on the dorsal aspect of the glans at approximately 12 weeks of gestation. After sequential proximal to distal remodeling of the ventral urethral plate along the ventral aspect of glans, the prepuce of epidermal origin fuses in the ventral midline.

Development of the human penis and clitoris.

The human penis and clitoris develop from the ambisexual genital tubercle. To compare and contrast the development of human penis and clitoris, we used macroscopic photography, optical projection tomography, light sheet microscopy, scanning electron microscopy, histology and immunohistochemistry. The human genital tubercle differentiates into a penis under the influence of androgens forming a tubular urethra that develops by canalization of the urethral plate to form a wide diamond-shaped urethral groove (opening zipper) whose edges (urethral folds) fuse in the midline (closing zipper). In contrast, in females, without the influence of androgens, the vestibular plate (homologue of the urethral plate) undergoes canalization to form a wide vestibular groove whose edges (vestibular folds) remain unfused, ultimately forming the labia minora defining the vaginal vestibule. The neurovascular anatomy is similar in both the developing human penis and clitoris and is the key to successful surgical reconstructions.

Contrasting mechanisms of penile urethral formation in mouse and human.

This paper addresses the developmental mechanisms of formation of the mouse and human penile urethra and the possibility that two disparate mechanisms are at play. It has been suggested that the entire penile urethra of the mouse forms via direct canalization of the endodermal urethral plate. While this mechanism surely accounts for development of the proximal portion of the mouse penile urethra, we suggest that the distal portion of the mouse penile urethra forms via a series of epithelial fusion events. Through review of the recent literature in combination with new data, it is unlikely that the entire mouse urethra is formed from the endodermal urethral plate due in part to the fact that from E14 onward the urethral plate is not present in the distal aspect of the genital tubercle. Formation of the distal portion of the mouse urethra receives substantial contribution from the preputial swellings that form the preputial-urethral groove and subsequently the preputial-urethral canal, the later of which is subdivided by a fusion event to form the distal portion of the mouse penile urethra. Examination of human penile development also reveals comparable dual morphogenetic mechanisms. However, in the case of human, direct canalization of the urethral plate occurs in the glans, while fusion events are involved in formation of the urethra within the penile shaft, a pattern exactly opposite to that of the mouse. The highest incidence of hypospadias in humans occurs at the junction of these two different developmental mechanisms. The relevance of the mouse as a model of human hypospadias is discussed.