Fast and slow muscle fiber transcriptome dynamics with lifelong endurance exercise.

We investigated fast and slow muscle fiber transcriptome exercise dynamics among three groups of men: lifelong exercisers (LLE, n = 8, 74 ± 1 yr), old healthy nonexercisers (OH, n = 9, 75 ± 1 yr), and young exercisers (YE, n = 8, 25 ± 1 yr). On average, LLE had exercised ∼4 day·wk-1 for ∼8 h·wk-1 over 53 ± 2 years. Muscle biopsies were obtained pre- and 4 h postresistance exercise (3 × 10 knee extensions at 70% 1-RM). Fast and slow fiber size and function were assessed preexercise with fast and slow RNA-seq profiles examined pre- and postexercise. LLE fast fiber size was similar to OH, which was ∼30% smaller than YE (P < 0.05) with contractile function variables among groups, resulting in lower power in LLE (P < 0.05). LLE slow fibers were ∼30% larger and more powerful compared with YE and OH (P < 0.05). At the transcriptome level, fast fibers were more responsive to resistance exercise compared with slow fibers among all three cohorts (P < 0.05). Exercise induced a comprehensive biological response in fast fibers (P < 0.05) including transcription, signaling, skeletal muscle cell differentiation, and metabolism with vast differences among the groups. Fast fibers from YE exhibited a growth and metabolic signature, with LLE being primarily metabolic, and OH showing a strong stress-related response. In slow fibers, only LLE exhibited a biological response to exercise (P < 0.05), which was related to ketone and lipid metabolism. The divergent exercise transcriptome signatures provide novel insight into the molecular regulation in fast and slow fibers with age and exercise and suggest that the ∼5% weekly exercise time commitment of the lifelong exercisers provided a powerful investment for fast and slow muscle fiber metabolic health at the molecular level.NEW & NOTEWORTHY This study provides the first insights into fast and slow muscle fiber transcriptome dynamics with lifelong endurance exercise. The fast fibers were more responsive to exercise with divergent transcriptome signatures among young exercisers (growth and metabolic), lifelong exercisers (metabolic), and old healthy nonexercisers (stress). Only lifelong exercisers had a biological response in slow fibers (metabolic). These data provide novel insights into fast and slow muscle fiber health at the molecular level with age and exercise.

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.

Mapping disease regulatory circuits at cell-type resolution from single-cell multiomics data.

Resolving chromatin-remodeling-linked gene expression changes at cell-type resolution is important for understanding disease states. Here we describe MAGICAL (Multiome Accessibility Gene Integration Calling and Looping), a hierarchical Bayesian approach that leverages paired single-cell RNA sequencing and single-cell transposase-accessible chromatin sequencing from different conditions to map disease-associated transcription factors, chromatin sites, and genes as regulatory circuits. By simultaneously modeling signal variation across cells and conditions in both omics data types, MAGICAL achieved high accuracy on circuit inference. We applied MAGICAL to study Staphylococcus aureus sepsis from peripheral blood mononuclear single-cell data that we generated from subjects with bloodstream infection and uninfected controls. MAGICAL identified sepsis-associated regulatory circuits predominantly in CD14 monocytes, known to be activated by bacterial sepsis. We addressed the challenging problem of distinguishing host regulatory circuit responses to methicillin-resistant and methicillin-susceptible S. aureus infections. Although differential expression analysis failed to show predictive value, MAGICAL identified epigenetic circuit biomarkers that distinguished methicillin-resistant from methicillin-susceptible S. aureus infections.

Deep phenotyping of the lipidomic response in COVID-19 and non-COVID-19 sepsis.

BACKGROUND: Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs-CoV-2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). METHODS: Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. RESULTS: Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID-19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α-III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12-HETE, exhibited relative specificity for COVID-19 and correlated with sPLA2 and the interleukin-13 receptor to drive lymphopenia, a marker of disease severity. Pro-inflammatory eicosanoids remained correlated with severity in COVID-19 28 days after admission. Amongst non-COVID ICU patients, elevations in 5- and 15-HETE and 9- and 13-HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflected disease severity in COVID-19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non-B non-T cell activation. In COVID-19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids - ChoE 18:3, LPC-O-16:0 and PC-O-30:0 - were altered specifically in COVID. LPC-O-16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi-omic inflammatory pathways and disease severity. CONCLUSIONS: A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12-HETE and the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients and correlate with the inflammatory response to link to disease severity.

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/.

Integrated single-cell multiome analysis reveals muscle fiber-type gene regulatory circuitry modulated by endurance exercise.

Endurance exercise is an important health modifier. We studied cell-type specific adaptations of human skeletal muscle to acute endurance exercise using single-nucleus (sn) multiome sequencing in human vastus lateralis samples collected before and 3.5 hours after 40 min exercise at 70% VO2max in four subjects, as well as in matched time of day samples from two supine resting circadian controls. High quality same-cell RNA-seq and ATAC-seq data were obtained from 37,154 nuclei comprising 14 cell types. Among muscle fiber types, both shared and fiber-type specific regulatory programs were identified. Single-cell circuit analysis identified distinct adaptations in fast, slow and intermediate fibers as well as LUM-expressing FAP cells, involving a total of 328 transcription factors (TFs) acting at altered accessibility sites regulating 2,025 genes. These data and circuit mapping provide single-cell insight into the processes underlying tissue and metabolic remodeling responses to exercise.

Deep Phenotyping of the Lipidomic Response in COVID and non-COVID Sepsis.

Lipids may influence cellular penetrance by pathogens and the immune response that they evoke. Here we find a broad based lipidomic storm driven predominantly by secretory (s) phospholipase A 2 (sPLA 2 ) dependent eicosanoid production occurs in patients with sepsis of viral and bacterial origin and relates to disease severity in COVID-19. Elevations in the cyclooxygenase (COX) products of arachidonic acid (AA), PGD 2 and PGI 2 , and the AA lipoxygenase (LOX) product, 12-HETE, and a reduction in the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients, correlate with the inflammatory response and link to disease severity. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflect disease severity in COVID-19. AA and LA metabolites and LPC-O-16:0 linked variably to the immune response. These studies yield prognostic biomarkers and therapeutic targets for patients with sepsis, including COVID-19. An interactive purpose built interactive network analysis tool was developed, allowing the community to interrogate connections across these multiomic data and generate novel hypotheses.

Human Immune Cell Epigenomic Signatures in Response to Infectious Diseases and Chemical Exposures.

Variations in DNA methylation patterns in human tissues have been linked to various environmental exposures and infections. Here, we identified the DNA methylation signatures associated with multiple exposures in nine major immune cell types derived from peripheral blood mononuclear cells (PBMCs) at single-cell resolution. We performed methylome sequencing on 111,180 immune cells obtained from 112 individuals who were exposed to different viruses, bacteria, or chemicals. Our analysis revealed 790,662 differentially methylated regions (DMRs) associated with these exposures, which are mostly individual CpG sites. Additionally, we integrated methylation and ATAC-seq data from same samples and found strong correlations between the two modalities. However, the epigenomic remodeling in these two modalities are complementary. Finally, we identified the minimum set of DMRs that can predict exposures. Overall, our study provides the first comprehensive dataset of single immune cell methylation profiles, along with unique methylation biomarkers for various biological and chemical exposures.

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.

Blood RNA alternative splicing events as diagnostic biomarkers for infectious disease.

Assays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62 = 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.

A methylation clock model of mild SARS-CoV-2 infection provides insight into immune dysregulation.

DNA methylation comprises a cumulative record of lifetime exposures superimposed on genetically determined markers. Little is known about methylation dynamics in humans following an acute perturbation, such as infection. We characterized the temporal trajectory of blood epigenetic remodeling in 133 participants in a prospective study of young adults before, during, and after asymptomatic and mildly symptomatic SARS-CoV-2 infection. The differential methylation caused by asymptomatic or mildly symptomatic infections was indistinguishable. While differential gene expression largely returned to baseline levels after the virus became undetectable, some differentially methylated sites persisted for months of follow-up, with a pattern resembling autoimmune or inflammatory disease. We leveraged these responses to construct methylation-based machine learning models that distinguished samples from pre-, during-, and postinfection time periods, and quantitatively predicted the time since infection. The clinical trajectory in the young adults and in a diverse cohort with more severe outcomes was predicted by the similarity of methylation before or early after SARS-CoV-2 infection to the model-defined postinfection state. Unlike the phenomenon of trained immunity, the postacute SARS-CoV-2 epigenetic landscape we identify is antiprotective.

Diverse effector and regulatory functions of fibro/adipogenic progenitors during skeletal muscle fibrosis in muscular dystrophy.

Fibrosis is a prominent pathological feature of skeletal muscle in Duchenne muscular dystrophy (DMD). The commonly used disease mouse model, mdx 5cv , displays progressive fibrosis in the diaphragm but not limb muscles. We use single-cell RNA sequencing to determine the cellular expression of the genes involved in extracellular matrix (ECM) production and degradation in the mdx 5cv diaphragm and quadriceps. We find that fibro/adipogenic progenitors (FAPs) are not only the primary source of ECM but also the predominant cells that express important ECM regulatory genes, including Ccn2, Ltbp4, Mmp2, Mmp14, Timp1, Timp2, and Loxs. The effector and regulatory functions are exerted by diverse FAP clusters which are different between diaphragm and quadriceps, indicating their activation by different tissue microenvironments. FAPs are more abundant in diaphragm than in quadriceps. Our findings suggest that the development of anti-fibrotic therapy for DMD should target not only the ECM production but also the pro-fibrogenic regulatory functions of FAPs.

Benchmarking transcriptional host response signatures for infection diagnosis.

Identification of host transcriptional response signatures has emerged as a new paradigm for infection diagnosis. For clinical applications, signatures must robustly detect the pathogen of interest without cross-reacting with unintended conditions. To evaluate the performance of infectious disease signatures, we developed a framework that includes a compendium of 17,105 transcriptional profiles capturing infectious and non-infectious conditions and a standardized methodology to assess robustness and cross-reactivity. Applied to 30 published signatures of infection, the analysis showed that signatures were generally robust in detecting viral and bacterial infections in independent data. Asymptomatic and chronic infections were also detectable, albeit with decreased performance. However, many signatures were cross-reactive with unintended infections and aging. In general, we found robustness and cross-reactivity to be conflicting objectives, and we identified signature properties associated with this trade-off. The data compendium and evaluation framework developed here provide a foundation for the development of signatures for clinical application. A record of this paper's transparent peer review process is included in the supplemental information.

Multi-objective optimization identifies a specific and interpretable COVID-19 host response signature.

The identification of a COVID-19 host response signature in blood can increase the understanding of SARS-CoV-2 pathogenesis and improve diagnostic tools. Applying a multi-objective optimization framework to both massive public and new multi-omics data, we identified a COVID-19 signature regulated at both transcriptional and epigenetic levels. We validated the signature's robustness in multiple independent COVID-19 cohorts. Using public data from 8,630 subjects and 53 conditions, we demonstrated no cross-reactivity with other viral and bacterial infections, COVID-19 comorbidities, or confounders. In contrast, previously reported COVID-19 signatures were associated with significant cross-reactivity. The signature's interpretation, based on cell-type deconvolution and single-cell data analysis, revealed prominent yet complementary roles for plasmablasts and memory T cells. Although the signal from plasmablasts mediated COVID-19 detection, the signal from memory T cells controlled against cross-reactivity with other viral infections. This framework identified a robust, interpretable COVID-19 signature and is broadly applicable in other disease contexts. A record of this paper's transparent peer review process is included in the supplemental information.

Transcriptome signatures preceding the induction of anti-stalk antibodies elicited after universal influenza vaccination.

A phase 1 clinical trial to test the immunogenicity of a chimeric group 1 HA (cHA) universal influenza virus vaccine targeting the conserved stalk domain of the hemagglutinin of influenza viruses was carried out. Vaccination with adjuvanted-inactivated vaccines induced high anti-stalk antibody titers. We sought to identify gene expression signatures that correlate with such induction. Messenger-RNA sequencing in whole blood was performed on the peripheral blood of 53 vaccinees. We generated longitudinal data on the peripheral blood of 53 volunteers, at early (days 3 and 7) and late (28 days) time points after priming and boosting with cHAs. Differentially expressed gene analysis showed no differences between placebo and live-attenuated vaccine groups. However, an upregulation of genes involved in innate immune responses and type I interferon signaling was found at day 3 after vaccination with inactivated adjuvanted formulations. Cell type deconvolution analysis revealed a significant enrichment for monocyte markers and different subsets of dendritic cells as mediators for optimal B cell responses and significant increase of anti-stalk antibodies in sera. A significant upregulation of immunoglobulin-related genes was only observed after administration of adjuvanted vaccines (either as primer or booster) with specific induction of anti-stalk IGVH1-69. This approach informed of specific immune signatures that correlate with robust anti-stalk antibody responses, while also helping to understand the regulation of gene expression induced by cHA proteins under different vaccine regimens.

Serum Fc-Mediated Monocyte Phagocytosis Activity Is Stable for Several Months after SARS-CoV-2 Asymptomatic and Mildly Symptomatic Infection.

We investigated the temporal profile of multiple components of the serological response after asymptomatic or mildly symptomatic SARS-CoV-2 infection, in a cohort of 67 previously SARS-CoV-2 naive young adults, up to 8.5 months after infection. We found a significant decrease of spike IgG and neutralization antibody titers from early (11 to 56 days) to late (4 to 8.5 months) time points postinfection. Over the study period, S1-specific IgG levels declined significantly faster than that of the S2-specific IgG. Further, serum antibodies from PCR-confirmed participants cross-recognized S2, but not S1, of the betacoronaviruses HKU1 and OC43, suggesting a greater degree of cross-reactivity of S2 among betacoronaviruses. Antibody-Dependent Natural Killer cell Activation (ADNKA) was detected at the early time point but significantly decreased at the late time point. Induction of serum Antibody-Dependent Monocyte Phagocytosis (ADMP) was detected in all the infected participants, and its levels remained stable over time. Additionally, a reduced percentage of participants had detectable neutralizing activity against the Beta (50%), Gamma (61 to 67%), and Delta (90 to 94%) variants, both early and late postinfection, compared to the ancestral strain (100%). Antibody binding to S1 and RBD of Beta, Gamma, Delta (1.7 to 2.3-fold decrease), and Omicron (10 to 16-fold decrease) variants was also significantly reduced compared to the ancestral SARS-CoV-2 strain. Overall, we found variable temporal profiles of specific components and functionality of the serological response to SARS-CoV-2 in young adults, which is characterized by lasting, but decreased, neutralizing activity and antibody binding to S1, stable ADMP activity, and relatively stable S2-specific IgG levels. IMPORTANCE Adaptive immunity mediated by antibodies is important for controlling SARS-CoV-2 infection. While vaccines against COVID-19 are currently widely distributed, a high proportion of the global population is still unvaccinated. Therefore, understanding the dynamics and maintenance of the naive humoral immune response to SARS-CoV-2 is of great importance. In addition, long-term responses after asymptomatic infection are not well-characterized, given the challenges in identifying such cases. Here, we investigated the longitudinal humoral profile in a well-characterized cohort of young adults with documented asymptomatic or mildly symptomatic SARS-CoV-2 infection. By analyzing samples collected preinfection, early after infection and during late convalescence, we found that, while neutralizing activity decreased over time, high levels of serum S2 IgG and Antibody-Dependent Monocyte Phagocytosis (ADMP) activity were maintained up to 8.5 months after infection. This suggests that a subset of antibodies with specific functions could contribute to long-term protection against SARS-CoV-2 in convalescent unvaccinated individuals.

Pre-infection antiviral innate immunity contributes to sex differences in SARS-CoV-2 infection.

Male sex is a major risk factor for SARS-CoV-2 infection severity. To understand the basis for this sex difference, we studied SARS-CoV-2 infection in a young adult cohort of United States Marine recruits. Among 2,641 male and 244 female unvaccinated and seronegative recruits studied longitudinally, SARS-CoV-2 infections occurred in 1,033 males and 137 females. We identified sex differences in symptoms, viral load, blood transcriptome, RNA splicing, and proteomic signatures. Females had higher pre-infection expression of antiviral interferon-stimulated gene (ISG) programs. Causal mediation analysis implicated ISG differences in number of symptoms, levels of ISGs, and differential splicing of CD45 lymphocyte phosphatase during infection. Our results indicate that the antiviral innate immunity set point causally contributes to sex differences in response to SARS-CoV-2 infection. A record of this paper's transparent peer review process is included in the supplemental information.

Asymptomatic or symptomatic SARS-CoV-2 infection plus vaccination confers increased adaptive immunity to variants of concern.

The ongoing evolution of SARS-CoV-2 requires monitoring the capability of immune responses to cross-recognize Variants of Concern (VOC). In this cross-sectional study, we examined serological and cell-mediated immune memory to SARS-CoV-2 variants, including Omicron, among a cohort of 18-21-year-old Marines with a history of either asymptomatic or mild SARS-CoV-2 infection 6 to 14 months earlier. Among the 210 participants in the study, 169 were unvaccinated while 41 received 2 doses of mRNA-based COVID-19 vaccines. Vaccination of previously infected participants strongly boosted neutralizing and binding activity and memory B and T cell responses including the recognition of Omicron, compared to infected but unvaccinated participants. Additionally, no measurable differences were observed in immune memory in healthy young adults with previous symptomatic or asymptomatic infections, for ancestral or variant strains. These results provide mechanistic immunological insights into population-based differences observed in immunity against Omicron and other variants among individuals with different clinical histories.

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.