Protein homeostasis depends on a delicate interplay between folding and degradation, orchestrated by molecular chaperones. Among them, Hsp90 is a central hub, regulating nearly 10% of the proteome through ATP-driven conformational cycles and selective interactions with cochaperones. The glucocorticoid receptor (GR) represents a paradigmatic Hsp90 client, whose maturation requires sequential remodeling steps involving multi-protein assemblies. While cryo-EM provided snapshots of these complexes, the dynamic determinants of GR activation and the antagonistic roles of cochaperones FKBP51 and FKBP52 remain poorly understood. Here, we integrate unbiased equilibrium atomistic molecular dynamics with nonequilibrium simulations of four different Hsp90-cochaperone-client assemblies that oversee distinct steps of GR maturation to elucidate how finely tuned dynamics and coordination/communication mechanisms determine functional emergence. Perturbations encoded by ligand insertion or removal reveal steroid binding as critical for both structural stability and inter-component communication. Ligand engagement not only stabilizes GR's active conformation but also feeds back to reshape chaperone and cochaperone dynamics, thereby modulating progression through the folding pathway. Steroid binding reinforces the interface in the Hsp90-p23-GR assembly, positioning cochaperone p23 as a molecular sensor for ligand occupancy. Comparative analyses of post-maturation complexes further uncover how immunophilins FKBP51 and FKBP52, despite structural similarity, elicit divergent allosteric effects on GR conformation and Hsp90-ATPase, determining opposing client fates. Our results establish ligand binding as an active modulator of chaperone-mediated folding, linking metabolic cues (ligand presence and levels) to client maturation. More broadly, they highlight cochaperones as dynamic checkpoints that selectively bias client outcomes, revealing generalizable principles of proteostasis regulation and opportunities for therapeutic intervention.

Dexamethasone sensitizes lens epithelial cells to ferroptosis through the glucocorticoid receptor/nuclear factor erythroid 2-related factor 2 signaling axis.

Prenatally elevated glucocorticoid disrupts social behavior via GR-Id3/E47-dependent astroglial dysfunction in LPS-induced autism-like rats.

Wnt/β-catenin signaling pathway is a highly conserved developmental pathway. This pathway is also involved in colorectal cancer and thus its selective targeting to cancer cells, albeit the risk involved, can serve as a promising therapeutic approach. Glucocorticoid receptor (GR) is a nuclear hormone receptor present in both cancer and non-cancer cells. Previously, we showed that cancer cell-associated GR, without eliciting any effect in normal cells, could be targeted for selective drug-sensitization in cancer cells. Based on this unique feature, we intended to sensitize the wnt/β-catenin pathway by co-formulating a GR-targeted cationic liposomal formulation carrying dexamethasone, a synthetic GR-ligand, and a wnt/β-catenin pathway inhibitor, FH535, to form D1XFH formulation. The nanometric and positively charged D1XFH formulation selectively kills colorectal cancer cells at much lower FH535 concentration than free drug or drug-associated GR-non-targeted liposome, while exhibiting unique nuclear uptake, increased ROS generation, apoptosis and G2-M phase cell cycle arrest in cancer cells. Further, in vivo data shows enhanced tumor-specific localization of this formulation, significant tumor growth inhibition and increased mice survivability, signifying its efficacy and biocompatibility in mouse colon subcutaneous and orthotopic tumor models. Protein expression analysis reveals enhanced reversal of epithelial-to-mesenchymal transition (EMT) and inhibition of various downstream proteins of wnt/β-catenin pathway. Additionally, analysis of tumor lysate from D1XFH-treated group shows an increased Th1/Th2 ratio, indicating favorable, anti-tumor immune response. The formulation exhibits no sub-chronic toxicity against healthy mice. In overall, our data strongly suggest that the GR-targeted FH535 liposomal delivery can safely target the highly sensitive wnt/β-catenin pathway for effective treatment of colorectal tumor.

This cross-sectional study investigated the relationship between depressive symptoms and creative personality through the lens of epigenetic modifications within Hypothalamic-Pituitary-Adrenal (HPA) axis genes. By analyzing DNA methylation patterns in key HPA axis genes-including corticotropin-releasing hormone (CRH), its receptor (CRHR1), CRH binding protein (CRHBP), melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (STAR), FK506 binding protein 5 (FKBP5), and the glucocorticoid receptor (NR3C1), we examined how methylation variation was associated with the depressive symptoms-creativity link in 371 participants (190 women, 181 men). Our findings revealed that among individuals with methylation patterns putatively associated with reduced HPA axis activity, higher depressive symptoms were positively associated with creative personality, suggesting that in this biological context, introspective aspects of depressive symptoms may coincide with creative propensities. Conversely, among individuals with methylation patterns putatively associated with heightened HPA axis activity, higher depressive symptoms were negatively associated with creative personality. These results suggest that the association between depressive symptoms and creativity is not uniform but differs based on methylation variation in genes involved in stress response regulation. The findings highlight the potential role of HPA axis-related biological factors in understanding individual differences in the depression-creativity relationship, though the cross-sectional design precludes causal inference.

Historically, it was thought that primary steroids released from endocrine glands exert their hormonal effects through corresponding receptors in peripheral tissues, and that their metabolism then inactivates them, followed by excretion. However, the metabolism of primary steroids is not just a way of inactivating and excreting them, but generates a variety of metabolites with different biological properties. In this review, we outline how various active steroid metabolites were discovered, describe some of the ways they are generated, and how they can in a non-classical way act on receptors or alter the activity of steroid metabolizing enzymes, thereby indirectly affecting receptor activities. Examples include the 5α-reduced ring-A metabolites of 11-deoxycorticosterone (DOC) and progesterone that are formed in the brain, act as neurosteroids and exert effects through the GABA-A membrane receptor. Another example is 11-ketoprogesterone that potently activates mineralocorticoid receptors (MR), but not glucocorticoid receptors (GR), and is more potent than its 11β-hydroxylated form, in contrast to glucocorticoids. Moreover, we discuss the microbiome as important source of bioactive metabolites, exemplified by the 11β-hydroxylated 5α-reduced ring-A corticosteroid and progesterone metabolites that were shown as potent 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) inhibitors. 11β-HSD2 inhibition results in cortisol-induced MR activation, sodium retention and hypertension. Furthermore, microbial 17,20-desmolase activity can convert glucocorticoids to androgens, potentially influencing diseases and therapeutic outcomes. There are still many knowledge gaps regarding bioactive steroid metabolites. Identifying additional bioactive steroid metabolites and characterizing their genomic and non-genomic effects should help uncovering their cell-specific functions and contributions to the maintenance of homeostatic regulation.

Central pathophysiological mechanisms underlying cognitive impairment and mood disorders are complex. Traditional Chinese Medicine (TCM)-derived bioactive compounds have significant research value in this field. This study aimed to synthesize current preclinical and emerging clinical evidence on the neuroprotective and psychotropic effects of key TCM constituents, with a particular focus on their roles in modulating neuroinflammatory signalling, synaptic plasticity, oxidative balance and stress-related neuroendocrine pathways. A narrative synthesis of experimental and early clinical studies was conducted, emphasizing mechanistic investigations in rodent models and exploratory human trials. Outcomes of interest included inflammatory cytokine expression, inflammasome activation, redox homeostasis, synaptic signalling pathways, neuroendocrine regulation, behavioural performance and translational pharmaceutical considerations. Multiple TCM constituents attenuate microglial activation and inflammasome signalling, suppressing interleukin-1β, interleukin-6 and tumor necrosis factor-alpha through inhibition of nuclear factor κB and NOD-like receptor pyrin domain-containing 3 pathways. These effects restore redox homeostasis, reduce synaptic loss and improve cognitive and behavioural outcomes in animal models. Concurrently, several compounds enhance synaptic resilience by upregulating brain-derived neurotrophic factor and tropomyosin receptor kinase B signalling, activating downstream mechanistic target of rapamycin complex 1 and cyclic adenosine monophosphate response element-binding protein pathways and preserving synaptic proteins. Key agents, including ginsenosides, baicalin and curcumin, have shown translational promise, with small human trials reporting improvements in depressive symptoms, cognitive function and biomarker profiles. Additionally, TCM compounds modulate HPA axis dynamics by attenuating stress-induced corticosterone elevation, restoring glucocorticoid receptor sensitivity and rebalancing monoaminergic and glutamatergic neurotransmission. However, pharmaceutical translation remains limited by challenges related to formulation, dosage standardization and poor oral bioavailability, particularly for flavonoids and saponins. TCM-derived compounds exert multifaceted neuroprotective and psychotropic effects, while successful clinical translation requires strengthened pharmaceutical characterization, standardized dosing strategies and advanced delivery systems such as nanoformulations, phytosomes and standardized granules to enhance bioavailability, reliability and regulatory acceptance.

Maternal physical activity enhances offspring metabolic resilience, whereas prenatal psychosocial stress predisposes offspring to lifelong metabolic dysfunction. Whether maternal exercise can attenuate the deleterious programming effects of stress has remained unclear. Here, we investigated the effect of maternal exercise and/or prenatal psychosocial stress on the metabolic health of male and female offspring. C57BL/6 dams were assigned to four conditions: sedentary/non-stressed (SED-NS), sedentary/stressed (SED-S), exercised/non-stressed (EX-NS), or exercised/stressed (EX-S). Male EX-NS offspring had improved glucose tolerance compared to SED-NS and SED-S; however these effects were blunted in male EX-S offspring, indicating that prenatal stress abolished this exercise-induced benefit. In contrast, there was no effect of maternal stress or exercise on glucose tolerance in female offspring. Maternal exercise chronically elevated basal corticosterone in male offspring, regardless of maternal stress. Maternal stress increased mineralocorticoid receptor (Nr3c2) mRNA and glucocorticoid receptor (NR3C1) protein expression in brown adipose tissue in male mice, while exercise restored NR3C1 expression. These findings indicate that prenatal stress blunts the metabolic benefits of maternal exercise in male offspring and is associated with altered corticosteroid signaling in brown adipose tissue. Together, the data identify a stress-exercise interaction that may influence offspring metabolic programming through tissue-specific regulation of glucocorticoid pathways.

The application of human hepatic cell lines to early drug discovery and development instead of human primary hepatocytes (HPHs) has been limited because of the low level of drug-metabolizing enzymes (DMEs). The study aimed to evaluate the effects of dexamethasone (DEX) treatment on DME expression, activities, and regulation in HuH-7 hepatoma cells. Expression and transcriptional regulation of major CYPs and UGTs in HuH-7 cells was evaluated by immunoblotting, probe substrate assays, and treatments with nuclear receptor agonists, including DEX, and antagonists. DEX increased the expression and activity of cytochrome P450 (CYP) 3A4, uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A1, and UGT2B7 but had minimal effects on CYP1A2, CYP2B6, or CYP2C9. These augmented activities of CYP3A4, UGT1A1, and UGT2B7 were concentration-dependently inhibited by their corresponding selective inhibitors. DEX-induced upregulation of CYP3A4 protein expression was abolished by co-treatment with the glucocorticoid receptor (GR) inhibitor, but not by co-treatment with the pregnane X receptor (PXR) inhibitor. However, treatment with PXR or constitutive androstane receptor (CAR) agonist did not lead to transcriptional activation of CYP3A4 and 2B6. Only CYP1A1 was transactivated by an aryl hydrocarbon receptor (AhR) ligand. Our results suggest that the activities of some major DMEs (CYP3A4, UGT1A1, and UGT2B7) in HuH-7 cells are promoted by DEX treatment possibly through GR activation. However, unlike HPHs, HuH-7 cells fail to show transcriptional regulation of DMEs by PXR or CAR, which limits their suitability for evaluating DME induction potential of investigational drugs.

The discovery of pathogenic variants in AKR1C1 and AKR1C2 in ultra-rare familial lipedema highlights steroid hormone metabolism as a core mechanism affecting about 11% of women during reproductive age. Lipedema represents a complex disease shaped by the interplay between rare mutations, common regulatory variants, and environmental exposures. This review outlines how ultra-rare monogenic mutations can illuminate the genetic and environmental bases of multifactorial lipedema. A systematic literature review (2000-2025) was performed using PubMed, Web of Science, and Google Scholar. Studies were identified with MeSH and keyword searches including lipedema, AKR1C1, AKR1C2, steroid metabolism, adipose tissue, obesogens, epigenetics, polycyclic aromatic hydrocarbons, endocrine disrupting chemicals, air pollution, and dietary hormones. The AKR1C1 p.Leu213Gln loss-of-function variant decreases progesterone inactivation by ~50% due to catalytic domain destabilization, leading to local progesterone accumulation. AKR1C2 gain-of-function variants and overexpression, found in 24% of cases, enhance DHT inactivation, converting it to 3α-androstanediol and suppressing anti-adipogenic androgen signaling. Population screening revealed three AKR1C1 polymorphisms associated with increased lipedema risk. The AKR1C2 regulatory variant rs28571848 in a glucocorticoid receptor site elevates AKR1C2/AKR1C3 expression and trunk fat mass independently of BMI. Environmental agents such as polycyclic aromatic hydrocarbons activate AKR1C1 via Nrf2-ARE signaling (3-10-fold induction), while steroid hormones promote adipocyte differentiation. Lipedema arises from an interaction between genetic susceptibility and environmental factors. Understanding the AKR1C pathway clarifies how genetic variants and obesogens disrupt steroid metabolism and induce epigenetic reprogramming, leading to clinical manifestations.

Overall survival with relacorilant and nab-paclitaxel in patients with platinum-resistant ovarian cancer (ROSELLA): a phase 3 randomised controlled trial.

Dexamethasone attenuates TNF-α-induced ototoxicity via Peroxiredoxin 6 upregulation in murine auditory hair cells and cochlea of noise-exposed mice.

Glucocorticoid-induced hypertension affects over 30% of treated patients, yet its underlying mechanisms remain unclear, particularly how glucocorticoids regulate renin within the renin-angiotensin-aldosterone system (RAAS). Modeling these dynamics is difficult because only four dose-response measurements are available at a single 24-h timepoint (36 observations total), while the system depends on roughly eleven biochemical parameters spanning minutes-long receptor interactions to days-long protein secretion. Classical parameter estimation becomes unreliable in this extremely underdetermined setting, and purely data-driven methods offer limited biological interpretability. In this paper, we introduce a physics-informed neural network (PINN) framework that integrates ELISA measurements from As4.1 juxtaglomerular cells, ordinary differential equations describing glucocorticoid receptor signaling and renin transcription, and automatic differentiation to enforce mechanistic constraints. By systematically tuning synthetic-data weights (SW in {0.2, 0.3, 0.5}), we identify an intermediate value of SW = 0.3 that provides the best overall balance between predictive accuracy, accepted ensemble size, and biologically plausible parameter estimates among the tested configurations. The framework uses adaptive constraint scheduling with a plateau ramp to reduce premature convergence and introduces calibrated plausibility thresholds reflecting experimental noise. The accepted PINN ensemble (n = 5, 50% success rate) achieved R2 = 0.803, compared with 0.759 for the SW = 0.5 baseline and −0.220 for the ODE-only baseline, with RMSE = 0.024. Key learned parameters (IC50 = 2.925 ± 0.012 mg/dL, Hill = 1.950 ± 0.009) are biologically plausible within the model assumptions, and the best single accepted model attained R2 = 0.891. Information criteria favored the PINN over the ODE model, with improvements of approximately 77× (AIC) and 5.9× (BIC). Despite training on a single timepoint, the PINN also infers full 48-h trajectories and reproduces non-monotonic dose-response behavior. This work presents, to our knowledge, the first PINN framework for glucocorticoid-mediated renin regulation and should be interpreted as a proof-of-concept approach for integrating sparse biomedical data with mechanistic constraints. The inferred parameters and temporal dynamics are best viewed as model-dependent, hypothesis-generating estimates rather than validated biological quantities.

Abstract: The Hypothalamic–Pituitary–Adrenal (HPA) axis dysfunction hypothesis of depression posits that maladaptive stress responsivity, sustained hypercortisolemia, and impaired Glucocorticoid Receptor (GR) signaling constitute core neurobiological mechanisms underlying depressive pathology. Dysregulation of this system, characterized by impaired glucocorticoid receptor feedback, sustained hypercortisolemia, and altered diurnal cortisol rhythms, contributes to neuronal vulnerability, maladaptive stress responsivity, and the progression of depressive pathology. This review synthesizes clinical and preclinical evidence linking HPA axis dysfunction to depression, with particular focus on key molecular targets including corticotropin-releasing hormone (CRH) receptors, glucocorticoid and mineralocorticoid receptors, and the arginine vasopressin system. In addition, we examine the emerging role of oxidative stress and inflammatory mediators in amplifying HPA dysregulation, and discuss the potential utility of biomarkers such as cortisol, ACTH, F2- isoprostanes, and 8-OHdG as diagnostic and treatment-responsive indicators. While current data provide compelling support for the HPA axis dysfunction hypothesis, methodological variability, reliance on peripheral measures, and limited longitudinal studies constrain causal inference. Future research should prioritize standardized biomarker panels, integration of multi-omics approaches, and validation of predictive signatures to enable precision medicine. Importantly, the review highlights novel therapeutic strategies, including CRH receptor antagonists, glucocorticoid receptor modulators, mineralocorticoid receptor agonists, and agents targeting oxidative and inflammatory pathways, as promising candidates for next-generation antidepressant development. By consolidating mechanistic, clinical, and translational evidence, this review highlights oxidative stress–HPA axis interactions as a pivotal determinant of depression vulnerability, underscores critical limitations in the current evidence base, and identifies future research priorities aimed at advancing biomarker-guided strategies for the prevention and treatment of depression.

Tumors foster an immunosuppressive microenvironment to evade the antitumor immune response. However, the influence of intratumoral immunosuppressive steroids on tumor-infiltrating natural killer (NK) cells and their implications for effective immunotherapy has remained largely unexplored. Here, we report that the functional enrichment of glucocorticoid cortisol signaling in the lung tumor microenvironment (TME) impairs NK cell anti-tumor cytotoxicity and exacerbates hypoxic stress. Cancer-associated fibroblasts (CAFs) and macrophages convert inactive cortisone to active cortisol, while T cells, fibroblasts, myeloid cells, macrophages, and cancer cells contribute to de novo steroid biosynthesis, collectively establishing a steroid-rich niche. Pharmacological inhibition of the glucocorticoid receptor (GR) in vivo alleviates cortisol-mediated immune suppression, resulting in reduced tumor growth and enhanced cytotoxicity of tumor-infiltrating NK cells. To overcome the cortisol-induced dysfunction of solid tumor targeting immunotherapy, we engineered chimeric antigen receptor (CAR) -NK cells specific to the Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) (highly expressed in lung tumors) and rendered them cortisol-resistant by genetic deletion of the cortisol receptor gene NR3C1. In cortisol-rich niches, cortisol-resistant CAR-NK cells sustained antitumor cytotoxicity. Mechanistically, NR3C1 deletion relieved cortisol-mediated suppression of PI3K-AKT-NF-κB signaling, restored anti-tumor activity, and markedly reduced hypoxic stress. In lung metastasis models, cortisol-resistant CAR-NK cells achieved superior tumor control and significantly reduced tumor burden compared with conventional CAR-NK cells. Together, these findings identify local cortisol signaling as a critical barrier to solid tumor immunotherapy and establish cortisol-resistant CAR-NK cells as a promising strategy for targeting steroidogenic solid tumors, which can be combined with therapeutic glucocorticoids.

Patients and physicians eagerly await the day when the adverse effects of glucocorticoids will no longer be considered a "necessary evil" in the treatment of inflammatory diseases. Research on novel GR ligands and combination therapies may bring us closer to this goal. We review the current status of novel approaches in rheumatology to reduce glucocorticoid toxicity, including the development of selective GR agonists and modulators (SEGRAMs), targeted delivery of glucocorticoid receptor ligands, and combination therapy of prednisone with an 11beta-HSD-1 inhibitor. We also describe advances in optimizing glucocorticoid use to achieve an optimal benefit-risk ratio when conventional glucocorticoids are necessary. A priority in the treatment of inflammatory rheumatic musculoskeletal diseases must be to avoid the use of glucocorticoids whenever possible and to use the lowest effective dose when there is no alternative. In this regard, we emphasize glucocorticoid-sparing concepts, for which new options have emerged in recent years. It is also important that glucocorticoid toxicity is carefully and fully documented in both clinical trials and practice in order to justify the development and use of alternative drugs that truly avoid glucocorticoid toxicity. Although we have made good progress in recent years toward the optimal use of glucocorticoids, there is still much to be done.

Nuclear receptors (NRs) comprise a superfamily of (ligand-)regulated transcription factors that are pivotal in orchestrating gene networks essential for development, metabolism, and cellular homeostasis. Their activity is critical for normal physiology, and consequently, dysregulation of NR signalling is implicated in a wide array of human diseases. Within this superfamily, the orphan nuclear receptor Nur77 and the glucocorticoid receptor (GR) are key regulators that exhibit significant cross-talk, primarily antagonistic, which is crucial for modulating inflammatory and stress responses. Despite the recognised importance of their interplay, the precise molecular mechanisms by which GR modulates Nur77's engagement with DNA remain incompletely defined. The present study elucidates the direct impact of GR and its ligand, dexamethasone (Dex), on the DNA binding dynamics of Nur77. Single-molecule DNA tightrope assays revealed that Nur77 employs a three-dimensional diffusion-based search mechanism on non-specific DNA, characterised by transient interactions with two distinct dissociation kinetic profiles. GR significantly stabilises Nur77-DNA interactions, evidenced by a shift towards longer residence times, primarily achieved by slowing the dissociation of the more transiently interacting Nur77 population. Conversely, single-molecule analysis and biochemical assays demonstrated that Dex alone markedly reduces Nur77's overall DNA binding affinity kinetics and frequency in a sequence-dependent manner, to such an extent that accurate quantification was unfeasible. These findings delineate distinct modulatory effects of the GR protein and its ligand on Nur77-DNA interactions, providing crucial biophysical insights into their complex regulatory interplay and revealing a direct, GR-independent impact of Dex on Nur77's DNA engagement.

Glucocorticoid receptor antagonists (GR antagonists) are a class of compounds developed to inhibit the activation of the glucocorticoid receptor and they have been used to treat a range of conditions such as Cushing's syndrome, diabetes, glaucoma, and depression. We report herein the discovery and optimization of a series of selective piperazine-based GR antagonists and discuss how key learnings from the discovery of relacorilant (CORT125134) were utilized to identify a simplified scaffold. Several compounds were identified with the desired profile and 3 key compounds were progressed to in vivo proof of concept studies.

The Developmental Origins of Health and Disease (DOHaD) hypothesis highlights the pivotal role of early-life nutrition in shaping lifelong health and disease risk. Low birth weight (LBW) remains a major public health issue associated with increased susceptibility to metabolic and cardiovascular disease, underscoring the need for early nutritional interventions. We investigated whether dietary supplementation with soy protein isolate (SPI) during lactation could mitigate adverse developmental programming in a rat model of LBW induced by maternal calorie restriction. Dams received an SPI-supplemented diet during lactation, and offspring were evaluated for postnatal growth, circulating IGF-1 and corticosterone concentrations, and pituitary expression of Gas5 lncRNA, miR-23b, and Pomc. Stress responsiveness and glucocorticoid receptor sensitivity were also assessed. SPI supplementation restored postnatal growth and IGF-1 concentrations in female offspring, and in males, it normalized pituitary Gas5 lncRNA and Pomc mRNA expressions, reduced stress-induced corticosterone hypersecretion, and improved pituitary glucocorticoid sensitivity. These findings indicate that SPI intervention during lactation can partially reverse epigenetic dysregulation of the stress and somatotropic axes caused by fetal undernutrition. Nutritional modulation during lactation thus represents a critical window for early intervention in LBW offspring. SPI supplementation may enhance endocrine and metabolic resilience, providing a practical nutritional programming approach to reduce future disease risk, consistent with the DOHaD paradigm.

Loss-of-function studies are a central approach to understanding gene/protein function. In mice, this often relies upon heritable recombination at the DNA level. This approach is slow and nonreversible, which limits both spatial and temporal resolution of analysis. Recently, degron techniques that directly target proteins for degradation have been successfully used to quickly and reversibly knock down proteins. Currently, these systems have been limited by lack of tissue/cell type specificity. Here, we generated mice that allow spatial and temporal control of GFP-tagged protein degradation. This DegronGFP line leads to degradation of GFP-tagged proteins in different cellular compartments and in distinct cell types. Further, it is rapid and reversible. We used DegronGFP to probe the function of the glucocorticoid receptor in the epidermis and demonstrate that it has distinct functions in proliferative and differentiated cells-an analysis that would not have been possible with traditional recombination approaches. We propose that the ability to use GFP knock-in lines for loss-of-function analysis will provide additional motivation for generation of these useful tools.