Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

The hypothalamic-pituitary-thyroid axis is intact in male insulin receptor substrate 4 knockout mice.

OBJECTIVE: Loss of function mutations in the insulin receptor substrate 4 (IRS4) gene cause a rare form of X-linked congenital central hypothyroidism in boys and men. Affected individuals show decreased thyroid-stimulation hormone (TSH) secretion. Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and downstream effectors. How loss of IRS4 affects TSH synthesis or secretion is unresolved. We therefore assessed IRS4's role in the hypothalamic-pituitary-thyroid axis of Irs4 knockout mice. METHODS: We generated two global Irs4 knockout mouse lines harboring either two or four base-pair deletions that result in frameshifts and loss of most of the IRS4 protein. RESULTS: Under normal laboratory conditions, Irs4 knockout males did not exhibit impairments in pituitary expression of TSH subunit genes (Tshb or Cga) or in the thyrotropin-releasing hormone (TRH) receptor. Additionally, their serum thyroid hormone, T3 (triiodothyronine) and T4 (thyroxine), and hypothalamic Trh expression levels were normal. When Irs4 knockouts were rendered hypothyroid with a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks, their serum TSH increased similarly to wild-type males. CONCLUSIONS: Overall, Irs4 knockout mice do not exhibit central hypothyroidism or otherwise appear to phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these animals.

Peak-agnostic high-resolution cis-regulatory circuitry mapping using single cell multiome data.

Single same cell RNAseq/ATACseq multiome data provide unparalleled potential to develop high resolution maps of the cell-type specific transcriptional regulatory circuitry underlying gene expression. We present CREMA, a framework that recovers the full cis-regulatory circuitry by modeling gene expression and chromatin activity in individual cells without peak-calling or cell type labeling constraints. We demonstrate that CREMA overcomes the limitations of existing methods that fail to identify about half of functional regulatory elements which are outside the called chromatin 'peaks'. These circuit sites outside called peaks are shown to be important cell type specific functional regulatory loci, sufficient to distinguish individual cell types. Analysis of mouse pituitary data identifies a Gata2-circuit for the gonadotrope-enriched disease-associated Pcsk1 gene, which is experimentally validated by reduced gonadotrope expression in a gonadotrope conditional Gata2-knockout model. We present a web accessible human immune cell regulatory circuit resource, and provide CREMA as an R package.

Automated single-cell omics end-to-end framework with data-driven batch inference.

To facilitate single cell multi-omics analysis and improve reproducibility, we present SPEEDI (Single-cell Pipeline for End to End Data Integration), a fully automated end-to-end framework for batch inference, data integration, and cell type labeling. SPEEDI introduces data-driven batch inference and transforms the often heterogeneous data matrices obtained from different samples into a uniformly annotated and integrated dataset. Without requiring user input, it automatically selects parameters and executes pre-processing, sample integration, and cell type mapping. It can also perform downstream analyses of differential signals between treatment conditions and gene functional modules. SPEEDI's data-driven batch inference method works with widely used integration and cell-typing tools. By developing data-driven batch inference, providing full end-to-end automation, and eliminating parameter selection, SPEEDI improves reproducibility and lowers the barrier to obtaining biological insight from these valuable single-cell datasets. The SPEEDI interactive web application can be accessed at https://speedi.princeton.edu/.

TSH Pulses Finely Tune Thyroid Hormone Release and TSH Receptor Transduction.

Detection of circulating TSH is a first-line test of thyroid dysfunction, a major health problem (affecting about 5% of the population) that, if untreated, can lead to a significant deterioration of quality of life and adverse effects on multiple organ systems. Human TSH levels display both pulsatile and (nonpulsatile) basal TSH secretion patterns; however, the importance of these in regulating thyroid function and their decoding by the thyroid is unknown. Here, we developed a novel ultra-sensitive ELISA that allows precise detection of TSH secretion patterns with minute resolution in mouse models of health and disease. We characterized the patterns of ultradian TSH pulses in healthy, freely behaving mice over the day-night cycle. Challenge of the thyroid axis with primary hypothyroidism because of iodine deficiency, a major cause of thyroid dysfunction worldwide, results in alterations of TSH pulsatility. Induction in mouse models of sequential TSH pulses that mimic ultradian TSH profiles in periods of minutes were more efficient than sustained rises in basal TSH levels at increasing both thyroid follicle cAMP levels, as monitored with a genetically encoded cAMP sensor, and circulating thyroid hormone. Hence, this mouse TSH assay provides a powerful tool to decipher how ultradian TSH pulses encode thyroid outcomes and to uncover hidden parameters in the TSH-thyroid hormone set-point in health and disease.

Activin E is a TGFß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming Growth Factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, similar to ActC. Collectively, our results establish ActE as an ALK7 ligand, thereby providing a link between genetic and in vivo studies of ActE as a regulator of adipose tissue.

Prerequisite endocardial-mesenchymal transition for murine cardiac trabecular angiogenesis.

Coronary heart disease damages the trabecular myocardium, and the regeneration of trabecular vessels may alleviate ischemic injury. However, the origins and developmental mechanisms of trabecular vessels remain unknown. Here, we show that murine ventricular endocardial cells generate trabecular vessels through an "angioEMT" mechanism. Time course fate mapping defined a specific wave of trabecular vascularization by ventricular endocardial cells. Single-cell transcriptomics and immunofluorescence identified a subpopulation of ventricular endocardial cells that underwent endocardial-mesenchymal transition (EMT) before these cells generated trabecular vessels. Ex vivo pharmacological activation and in vivo genetic inactivation experiments identified an EMT signal in ventricular endocardial cells involving SNAI2-TGFB2/TGFBR3, which was a prerequisite for later trabecular-vessel formation. Additional loss- and gain-of-function genetic studies showed that VEGFA-NOTCH1 signaling regulated post-EMT trabecular angiogenesis by ventricular endocardial cells. Our finding that trabecular vessels originate from ventricular endocardial cells through a two-step angioEMT mechanism could inform better regeneration medicine for coronary heart disease.

Activating Transcription Factor 3 Stimulates Follicle-Stimulating Hormone-ß Expression In Vitro But Is Dispensable for Follicle-Stimulating Hormone Production in Murine Gonadotropes In Vivo.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHß subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone ß (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LßT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.

The First Latin American Consensus on Superficial and Perforating Venous Mapping.

With the aim of obtaining a map which is useful as a diagnostic tool and therapeutical orientation, complementing the written report of duplex ultrasound venous study, Latin-American Scientific Societies of Phlebology, Vascular Surgery and Vascular Imaging were invited to participate, through their regional representatives, to the First Consensus of Superficial and Perforating Venous Mapping. A consensus process using a modified Delphi method was carried out. An International Working Group was formed, which developed a Prototype of the Venous Mapping that worked as a starting point for consensus, and was presented in a first virtual meeting of 54 experts (societies' representatives) when the methodology was explained. For the consensus process, two rounds of self-administrated questionnaires with feedback were used. In the first questionnaire a 100% consensus was obtained in the 15 statements (an agreement range of 85.2% to 100%) In the analysis of qualitative data, three categories according to the actions to implement were identified - actions which involved no action, minor changes and major changes. This analysis was used to build the second questionnaire, which reached a consensus in its six statements (agreement range of 87.1% to 98.1%). A final consensus on every field proposed was established with the approval of all the experts consulted and it was presented at a third online meeting. The document of the superficial and perforating venous mapping reached by consensus is presented hereafter.

High Prevalence of Alternative Diagnoses in Children and Adolescents with Suspected Long COVID-A Single Center Cohort Study.

BACKGROUND: Long COVID (LC) is a diagnosis that requires exclusion of alternative somatic and mental diseases. The aim of this study was to examine the prevalence of differential diagnoses in suspected pediatric LC patients and assess whether adult LC symptom clusters are applicable to pediatric patients. MATERIALS AND METHODS: Pediatric presentations at the Pediatric Infectious Diseases Department of the University Hospital Essen (Germany) were assessed retrospectively. The correlation of initial symptoms and final diagnoses (LC versus other diseases or unclarified) was assessed. The sensitivity, specificity, negative and positive predictive values of adult LC symptom clusters were calculated. RESULTS: Of 110 patients, 32 (29%) suffered from LC, 52 (47%) were diagnosed with alternative somatic/mental diseases, and 26 (23%) remained unclarified. Combined neurological and respiratory clusters displayed a sensitivity of 0.97 (95% CI 0.91-1.00) and a negative predictive value of 0.97 (0.92-1.00) for LC. DISCUSSION/CONCLUSIONS: The prevalence of alternative somatic and mental diseases in pediatric patients with suspected LC is high. The range of underlying diseases is wide, including chronic and potentially life-threatening conditions. Neurological and respiratory symptom clusters may help to identify patients that are unlikely to be suffering from LC.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Follistatin Forms a Stable Complex With Inhibin A That Does Not Interfere With Activin A Antagonism.

Inhibins are transforming growth factor-ß family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common ß chain with activin ligands. Follistatin is another activin antagonist, known to bind the common ß chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin ß chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lower affinity than follistatin 288 alone or in complex with activin A. Characterizing the inhibin A:follistatin 288 complex in an activin-responsive luciferase assay and by surface plasmon resonance indicated that the inhibitor complex readily dissociated upon binding type II receptor activin receptor type IIb, allowing both antagonists to inhibit activin signaling. Additionally, injection of the complex in ovariectomized female mice did not alter inhibin A suppression of FSH. Taken together, this study shows that while follistatin binds to inhibin A with a substochiometric ratio relative to the activin homodimer, the complex can dissociate readily, allowing both proteins to effectively antagonize activin signaling.

Steroidogenic Factor 1 Regulates Transcription of the Inhibin B Coreceptor in Pituitary Gonadotrope Cells.

The inhibins control reproduction by suppressing follicle-stimulating hormone synthesis in pituitary gonadotrope cells. The newly discovered inhibin B coreceptor, TGFBR3L, is selectively and highly expressed in gonadotropes in both mice and humans. Here, we describe our initial characterization of mechanisms controlling cell-specific Tgfbr3l/TGFBR3L transcription. We identified two steroidogenic factor 1 (SF-1 or NR5A1) cis-elements in the proximal Tgfbr3l promoter in mice. SF-1 induction of murine Tgfbr3l promoter-reporter activity was inhibited by mutations in one or both sites in heterologous cells. In homologous cells, mutation of these cis-elements or depletion of endogenous SF-1 similarly decreased reporter activity. We observed nearly identical results when using a human TGFBR3L promoter-reporter. The Tgfbr3l gene was tightly compacted and Tgfbr3l mRNA expression was essentially absent in gonadotropes of SF-1 (Nr5a1) conditional knockout mice. During murine embryonic development, Tgfbr3l precedes Nr5a1 expression, though the two transcripts are fully colocalized by embryonic day 18.5 and thereafter. Collectively, these data indicate that SF-1 directly regulates Tgfbr3l/TGFBR3L transcription and is required for postnatal expression of the gene in gonadotropes.

Transcription factor GATA2 may potentiate follicle-stimulating hormone production in mice via induction of the BMP antagonist gremlin in gonadotrope cells.

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHß subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

The extant immunoglobulin superfamily, member 1 gene results from an ancestral gene duplication in eutherian mammals.

Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein with high expression in the mammalian pituitary gland. Mutations in the IGSF1 gene cause congenital central hypothyroidism in humans. The IGSF1 protein is co-translationally cleaved into N- and C-terminal domains (NTD and CTD), the latter of which is trafficked to the plasma membrane and appears to be the functional portion of the molecule. Though the IGSF1-NTD is retained in the endoplasmic reticulum and has no apparent function, it has a high degree of sequence identity with the IGSF1-CTD and is conserved across mammalian species. Based upon phylogenetic analyses, we propose that the ancestral IGSF1 gene encoded the IGSF1-CTD, which was duplicated and integrated immediately upstream of itself, yielding a larger protein encompassing the IGSF1-NTD and IGSF1-CTD. The selective pressures favoring the initial gene duplication and subsequent retention of a conserved IGSF1-NTD are unresolved.

IGSF1 Deficiency Leads to Reduced TSH Production Independent of Alterations in Thyroid Hormone Action in Male Mice.

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action.

The orphan ligand, activin C, signals through activin receptor-like kinase 7.

Activin ligands are formed from two disulfide-linked inhibin ß (Inhß) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhßA/InhßA) or activin C (ActC; InhßC/InhßC), or as heterodimers, as with activin AC (ActAC; InhßA:InhßC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhßC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhßC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.

Knowledge, Attitudes, and Preparedness for Managing Pregnant and Postpartum Women with COVID-19 Among Nurse-Midwives in Kenya.

Introduction: Globally, maternal morbidity and mortality have increased during the COVID-19 pandemic. Given the high burden of maternal and neonatal mortality in Kenya prior to COVID-19, front line health workers, including nurse-midwives, must be competent to ensure continued quality maternal services. Knowledge and awareness of COVID-19 transmission influence nurse-midwives risk perception and ability to implement prevention strategies. Objective: We examined nurse-midwives' knowledge, attitudes, and preparedness in managing pregnant and postpartum women with COVID-19 in Kenya. Methods: A cross-sectional online survey was conducted among 118 nurse-midwives between July 2020 and November 2020. A 31-item survey comprising 15 knowledge, 11 attitude, and five preparedness questions was administered using SurveyMonkey. A link to the survey was distributed among nurse-midwives via email. Multiple logistic regression analysis was used to assess associations between the variables. A p-value <.05 was considered statistically significant. Results: Eighty-five participants were included in the final analysis (response rate 72%). Most participants were female (n = 69, 81.2%), 52.9% (n = 45) worked in labor wards, and 57.6% (n = 49) worked in rural hospitals. Overall, 71% (n = 57) of participants had sufficient knowledge about managing COVID-19 in pregnant and postpartum women. However, only 63% were willing to receive COVID-19 vaccination. Nurse-midwives working in urban areas were 3.7 times more likely to have positive attitudes than those in rural areas (odds ratio 3.724, 95% confidence interval 1.042-13.31; p = .043). Conclusion: Nurse-midwives' responses to the Kenyan government's COVID-19 guidelines for managing and caring for pregnant women were inconsistent. Continued professional development for nurse-midwives is important to ensure they stay abreast of evolving COVID-19 guidelines for maternal health. Our findings also suggest vaccine hesitancy may be a hurdle for ongoing COVID-19 vaccination.

Single nucleus transcriptome and chromatin accessibility of postmortem human pituitaries reveal diverse stem cell regulatory mechanisms.

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.