Reconstructing human brown fat developmental trajectory in vitro.

Brown adipocytes (BAs) represent a specialized cell type that is able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. In humans, the brown fat tissue is composed of discrete depots found throughout the neck and trunk region. BAs originate from a precursor common to skeletal muscle, but their developmental trajectory remains poorly understood. Here, we used single-cell RNA sequencing to characterize the development of interscapular brown fat in mice. Our analysis identified a transient stage of BA differentiation characterized by the expression of the transcription factor GATA6. We show that recapitulating the sequence of signaling cues identified in mice can lead to efficient differentiation of BAs in vitro from human pluripotent stem cells. These precursors can in turn be efficiently converted into functional BAs that can respond to signals mimicking adrenergic stimuli by increasing their metabolism, resulting in heat production.

Fibroblast-derived Hgf controls recruitment and expansion of muscle during morphogenesis of the mammalian diaphragm.

The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, congenital diaphragmatic hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors and effects on phrenic nerve have not been explicitly tested. Using conditional mutagenesis in mice and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue in the diaphragm, are the source of HGF critical for diaphragm muscularization. PPF-derived HGF is directly required for recruitment of MET+ muscle progenitors to the diaphragm and indirectly (via its effect on muscle development) required for phrenic nerve primary branching. In addition, HGF is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Localization of HGF at the diaphragm's leading edges directs dorsal and ventral expansion of muscle and regulates its overall size and shape. Surprisingly, large muscleless regions in HGF and Met mutants do not lead to hernias. While these regions are likely more susceptible to CDH, muscle loss is not sufficient to cause CDH.

Favipiravir for Treatment of Outpatients With Asymptomatic or Uncomplicated Coronavirus Disease 2019: A Double-Blind, Randomized, Placebo-Controlled, Phase 2 Trial.

BACKGROUND: Favipiravir, an oral, RNA-dependent RNA polymerase inhibitor, has in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite limited data, favipiravir is administered to patients with coronavirus disease 2019 (COVID-19) in several countries. METHODS: We conducted a phase 2, double-blind, randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV-2 reverse-transcription polymerase chain reaction assay (RT-PCR) within 72 hours of enrollment. Participants were randomized to receive placebo or favipiravir (1800 mg twice daily [BID] day 1, 800 mg BID days 2-10). The primary outcome was SARS-CoV-2 shedding cessation in a modified intention-to-treat (mITT) cohort of participants with positive enrollment RT-PCRs. Using SARS-CoV-2 amplicon-based sequencing, we assessed favipiravir's impact on mutagenesis. RESULTS: We randomized 149 participants with 116 included in the mITT cohort. The participants' mean age was 43 years (standard deviation, 12.5 years) and 57 (49%) were women. We found no difference in time to shedding cessation overall (hazard ratio [HR], 0.76 favoring placebo [95% confidence interval {CI}, .48-1.20]) or in subgroups (age, sex, high-risk comorbidities, seropositivity, or symptom duration at enrollment). We detected no difference in time to symptom resolution (initial: HR, 0.84 [95% CI, .54-1.29]; sustained: HR, 0.87 [95% CI, .52-1.45]) and no difference in transition mutation accumulation in the viral genome during treatment. CONCLUSIONS: Our data do not support favipiravir at commonly used doses in outpatients with uncomplicated COVID-19. Further research is needed to ascertain if higher favipiravir doses are effective and safe for patients with COVID-19. CLINICAL TRIALS REGISTRATION: NCT04346628.

Cell culture system to assay candidate genes and molecular pathways implicated in congenital diaphragmatic hernias.

The mammalian muscularized diaphragm is essential for respiration and defects in the developing diaphragm cause a common and frequently lethal birth defect, congenital diaphragmatic hernia (CDH). Human genetic studies have implicated more than 150 genes and multiple molecular pathways in CDH, but few of these have been validated because of the expense and time to generate mouse mutants. The pleuroperitoneal folds (PPFs) are transient embryonic structures in diaphragm development and defects in PPFs lead to CDH. We have developed a system to culture PPF fibroblasts from E12.5 mouse embryos and show that these fibroblasts, in contrast to the commonly used NIH 3T3 fibroblasts, maintain expression of key genes in normal diaphragm development. Using pharmacological and genetic manipulations that result in CDH in vivo, we also demonstrate that differences in proliferation provide a rapid means of distinguishing healthy and impaired PPF fibroblasts. Thus, the PPF fibroblast cell culture system is an efficient tool for assaying the functional significance of CDH candidate genes and molecular pathways and will be an important resource for elucidating the complex etiology of CDH.

Connecting muscle development, birth defects, and evolution: An essential role for muscle connective tissue.

Skeletal muscle powers all movement of the vertebrate body and is distributed in multiple regions that have evolved distinct functions. Axial muscles are ancestral muscles essential for support and locomotion of the whole body. The evolution of the head was accompanied by development of cranial muscles essential for eye movement, feeding, vocalization, and facial expression. With the evolution of paired fins and limbs and their associated muscles, vertebrates gained increased locomotor agility, populated the land, and acquired fine motor skills. Finally, unique muscles with specialized functions have evolved in some groups, and the diaphragm which solely evolved in mammals to increase respiratory capacity is one such example. The function of all these muscles requires their integration with the other components of the musculoskeletal system: muscle connective tissue (MCT), tendons, bones as well as nerves and vasculature. MCT is muscle's closest anatomical and functional partner. Not only is MCT critical in the adult for muscle structure and function, but recently MCT in the embryo has been found to be crucial for muscle development. In this review, we examine the important role of the MCT in axial, head, limb, and diaphragm muscles for regulating normal muscle development, discuss how defects in MCT-muscle interactions during development underlie the etiology of a range of birth defects, and explore how changes in MCT development or communication with muscle may have led to the modification and acquisition of new muscles during vertebrate evolution.

Unique morphogenetic signatures define mammalian neck muscles and associated connective tissues.

In vertebrates, head and trunk muscles develop from different mesodermal populations and are regulated by distinct genetic networks. Neck muscles at the head-trunk interface remain poorly defined due to their complex morphogenesis and dual mesodermal origins. Here, we use genetically modified mice to establish a 3D model that integrates regulatory genes, cell populations and morphogenetic events that define this transition zone. We show that the evolutionary conserved cucullaris-derived muscles originate from posterior cardiopharyngeal mesoderm, not lateral plate mesoderm, and we define new boundaries for neural crest and mesodermal contributions to neck connective tissue. Furthermore, lineage studies and functional analysis of Tbx1- and Pax3-null mice reveal a unique developmental program for somitic neck muscles that is distinct from that of somitic trunk muscles. Our findings unveil the embryological and developmental requirements underlying tetrapod neck myogenesis and provide a blueprint to investigate how muscle subsets are selectively affected in some human myopathies.

Developmental origin and morphogenesis of the diaphragm, an essential mammalian muscle.

The diaphragm is a mammalian skeletal muscle essential for respiration and for separating the thoracic and abdominal cavities. Development of the diaphragm requires the coordinated development of muscle, muscle connective tissue, tendon, nerves, and vasculature that derive from different embryonic sources. However, defects in diaphragm development are common and the cause of an often deadly birth defect, Congenital Diaphragmatic Hernia (CDH). Here we comprehensively describe the normal developmental origin and complex spatial-temporal relationship between the different developing tissues to form a functional diaphragm using a developmental series of mouse embryos genetically and immunofluorescently labeled and analyzed in whole mount. We find that the earliest developmental events are the emigration of muscle progenitors from cervical somites followed by the projection of phrenic nerve axons from the cervical neural tube. Muscle progenitors and phrenic nerve target the pleuroperitoneal folds (PPFs), transient pyramidal-shaped structures that form between the thoracic and abdominal cavities. Subsequently, the PPFs expand across the surface of the liver to give rise to the muscle connective tissue and central tendon, and the leading edge of their expansion precedes muscle morphogenesis, formation of the vascular network, and outgrowth and branching of the phrenic nerve. Thus development and morphogenesis of the PPFs is critical for diaphragm formation. In addition, our data indicate that the earliest events in diaphragm development are critical for the etiology of CDH and instrumental to the evolution of the diaphragm. CDH initiates prior to E12.5 in mouse and suggests that defects in the early PPF formation or their ability to recruit muscle are an important source of CDH. Also, the recruitment of muscle progenitors from cervical somites to the nascent PPFs is uniquely mammalian and a key developmental innovation essential for the evolution of the muscularized diaphragm.

A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle.

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ-organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

Homology of the cranial vault in birds: new insights based on embryonic fate-mapping and character analysis.

Bones of the cranial vault appear to be highly conserved among tetrapod vertebrates. Moreover, bones identified with the same name are assumed to be evolutionarily homologous. However, recent developmental studies reveal a key difference in the embryonic origin of cranial vault bones between representatives of two amniote lineages, mammals and birds, thereby challenging this view. In the mouse, the frontal is derived from cranial neural crest (CNC) but the parietal is derived from mesoderm, placing the CNC-mesoderm boundary at the suture between these bones. In the chicken, this boundary is located within the frontal. This difference and related data have led several recent authors to suggest that bones of the avian cranial vault are misidentified and should be renamed. To elucidate this apparent conflict, we fate-mapped CNC and mesoderm in axolotl to reveal the contributions of these two embryonic cell populations to the cranial vault in a urodele amphibian. The CNC-mesoderm boundary in axolotl is located between the frontal and parietal bones, as in the mouse but unlike the chicken. If, however, the avian frontal is regarded instead as a fused frontal and parietal (i.e. frontoparietal) and the parietal as a postparietal, then the cranial vault of birds becomes developmentally and topologically congruent with those of urodeles and mammals. This alternative hypothesis of cranial vault homology is also phylogenetically consistent with data from the tetrapod fossil record, where frontal, parietal and postparietal bones are present in stem lineages of all extant taxa, including birds. It further implies that a postparietal may be present in most non-avian archosaurs, but fused to the parietal or supraoccipital as in many extant mammals.

Evolution of the head-trunk interface in tetrapod vertebrates.

Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes.

Dual embryonic origin and patterning of the pharyngeal skeleton in the axolotl (Ambystoma mexicanum).

The impressive morphological diversification of vertebrates was achieved in part by innovation and modification of the pharyngeal skeleton. Extensive fate mapping in amniote models has revealed a primarily cranial neural crest derivation of the pharyngeal skeleton. Although comparable fate maps of amphibians produced over several decades have failed to document a neural crest derivation of ventromedial elements in these vertebrates, a recent report provides evidence of a mesodermal origin of one of these elements, basibranchial 2, in the axolotl. We used a transgenic labeling protocol and grafts of labeled cells between GFP+ and white embryos to derive a fate map that describes contributions of both cranial neural crest and mesoderm to the axolotl pharyngeal skeleton, and we conducted additional experiments that probe the mechanisms that underlie mesodermal patterning. Our fate map confirms a dual embryonic origin of the pharyngeal skeleton in urodeles, including derivation of basibranchial 2 from mesoderm closely associated with the second heart field. Additionally, heterotopic transplantation experiments reveal lineage restriction of mesodermal cells that contribute to pharyngeal cartilage. The mesoderm-derived component of the pharyngeal skeleton appears to be particularly sensitive to retinoic acid (RA): administration of exogenous RA leads to loss of the second basibranchial, but not the first. Neural crest was undoubtedly critical in the evolution of the vertebrate pharyngeal skeleton, but mesoderm may have played a central role in forming ventromedial elements, in particular. When and how many times during vertebrate phylogeny a mesodermal contribution to the pharyngeal skeleton evolved remain to be resolved.

A molecular mechanism for the origin of a key evolutionary innovation, the bird beak and palate, revealed by an integrative approach to major transitions in vertebrate history.

The avian beak is a key evolutionary innovation whose flexibility has permitted birds to diversify into a range of disparate ecological niches. We approached the problem of the mechanism behind this innovation using an approach bridging paleontology, comparative anatomy, and experimental developmental biology. First, we used fossil and extant data to show the beak is distinctive in consisting of fused premaxillae that are geometrically distinct from those of ancestral archosaurs. To elucidate underlying developmental mechanisms, we examined candidate gene expression domains in the embryonic face: the earlier frontonasal ectodermal zone (FEZ) and the later midfacial WNT-responsive region, in birds and several reptiles. This permitted the identification of an autapomorphic median gene expression region in Aves. To test the mechanism, we used inhibitors of both pathways to replicate in chicken the ancestral amniote expression. Altering the FEZ altered later WNT responsiveness to the ancestral pattern. Skeletal phenotypes from both types of experiments had premaxillae that clustered geometrically with ancestral fossil forms instead of beaked birds. The palatal region was also altered to a more ancestral phenotype. This is consistent with the fossil record and with the tight functional association of avian premaxillae and palate in forming a kinetic beak.

MK-2206, an AKT inhibitor, promotes caspase-independent cell death and inhibits leiomyoma growth.

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs.

The role of progesterone signaling in the pathogenesis of uterine leiomyoma.

Uterine leiomyomas are benign tumors that originate from the myometrium. Evidence points to ovarian steroid hormones, in particular, progesterone as major promoters of leiomyoma development and growth. While progesterone action in leiomyomas involves the classical nuclear receptor effects on gene regulation, there is growing evidence that signaling pathways are directly activated by the progesterone receptor (PR) and that PR can interact with growth factor signaling systems to promote proliferation and survival of leiomyomas. Studies investigating the genomic and non-genomic actions of PR and its role in leiomyoma growth are summarized here. Studies testing various selective progesterone receptor modulators for the treatment of leiomyomas are also highlighted. An increased understanding of the mechanisms associated with progesterone-driven growth of leiomyomas is critical in order to develop more efficient and targeted therapies for this prevalent disease.

Cyp26 enzymes function in endoderm to regulate pancreatic field size.

The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe (gir) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.

The role of genetic variation in the lamin a/c gene in the etiology of polycystic ovary syndrome.

OBJECTIVE: We performed this study to test the hypothesis that variation in the lamin a/c gene (LMNA) contributes to milder phenotypes of insulin resistance, hyperandrogenism, and/or metabolic syndrome associated with polycystic ovary syndrome (PCOS). RESEARCH DESIGN AND METHODS: We resequenced the coding region, flanking intronic, and proximal promoter regions of the lamin a/c gene in 43 women with PCOS with evidence of upper-body obesity (waist circumference >88 cm) and identified 56 variants, two of which were nonsynonymous substitutions (lmna11 exon1 E98D; lmna24 exon 7 R455C). We genotyped 53 single-nucleotide polymorphisms (44 identified through resequencing and nine included to maximize informativeness of the entire gene) in 624 index (PCOS) cases and 544 controls of European ancestry. We tested for association between these variants and PCOS. In a subset of individuals, we also tested for association with metabolic syndrome and quantitative traits (body mass index, waist circumference, total testosterone, dehydroepiandrosterone sulfate, fasting glucose and insulin, low-density lipoprotein, and total triglycerides). RESULTS: After correction for multiple testing, none of the variants showed significant evidence for association with PCOS, the metabolic syndrome, or any of the quantitative traits tested. CONCLUSIONS: Whereas these studies cannot exclude the role of genetic variation in the lamin a/c gene in isolated cases of PCOS, we can conclude that common variation in the lamin a/c gene does not contribute to the etiology of PCOS in women of European ancestry.

Progestins activate the AKT pathway in leiomyoma cells and promote survival.

CONTEXT: Progesterone has been associated with promoting growth of uterine leiomyomas. The mechanisms involved remain unclear. OBJECTIVE: In this study we investigated the activation of the AKT pathway and its downstream effectors, glycogen synthase kinase-3b and Forkhead box O (FOXO)-1 by progesterone as a mechanism of proliferation and survival of leiomyoma cells. Inhibitors of the AKT pathway were used to demonstrate the role of phosphatidylinositol 3-kinase, AKT, and FOXO1 in contributing to cell proliferation and apoptosis. RESULTS: Treatment of leiomyoma cells with R5020 over a period of 72 h resulted in higher cell numbers compared with untreated cells. When cells were treated with 100 nm R5020 for 1 and 24 h, the levels of phospho(Ser 473)-AKT increased. This increase was inhibited when cells were cotreated with RU486. Treatment of leiomyoma cells with a phosphatidylinositol 3-kinase inhibitor, LY294 dramatically decreased levels of phospho(Ser 473)-AKT, despite R5020 treatment. In addition to increased phospho(Ser 473)-AKT levels, R5020 treatment resulted in an increase in phospho(Ser 256)-FOXO1 and phosphoglycogen synthase kinase-3b. Inhibition of AKT using API-59 decreased proliferation and cell viability even in the presence of R5020. Higher concentrations of API-59-induced apoptosis of leiomyoma cells, even in the presence of R5020. Psammaplysene A increased nuclear FOXO1 levels and did not affect cell proliferation but induced apoptosis of leiomyoma cells. CONCLUSIONS: The progestin, R5020, can rapidly activate the AKT pathway. Inhibition of the AKT pathway inhibits cell proliferation and promotes apoptosis of leiomyoma cells.

Progesterone receptor action in leiomyoma and endometrial cancer.

Progesterone is a key hormone in the regulation of uterine function. In the normal physiological context, progesterone is primarily involved in remodeling of the endometrium and maintaining a quiescent myometrium. When pathologies of the uterus develop, specifically, endometrial cancer and uterine leiomyoma, response to progesterone is usually altered. Progesterone acts through mainly two isoforms of the progesterone receptor (PR), PRA and PRB which have been reported to exhibit different transcriptional activities. Studies examining the expression and function of the PRs in the normal endometrium and myometrium as well as in endometrial cancer and uterine leiomyoma are summarized here. The clinical use of progestins and the transcriptional activity of the PR on genes specific to endometrial cancer and leiomyoma are described. An increased understanding of the differential expression of PRs and response to progesterone in these two diseases is critical in order to develop more efficient and targeted therapies.