Co-expression of TNF receptors 1 and 2 on melanomas facilitates soluble TNF-induced resistance to MAPK pathway inhibitors.

BACKGROUND: The effectiveness of MAPK pathway inhibitors (MAPKi) used to treat patients with BRAF-mutant melanoma is limited by a range of resistance mechanisms, including soluble TNF (solTNF)-mediated NF-kB signaling. solTNF preferentially signals through type-1 TNF receptor (TNFR1), however, it can also bind to TNFR2, a receptor that is primarily expressed on leukocytes. Here, we investigate the TNFR2 expression pattern on human BRAFV600E+ melanomas and its role in solTNF-driven resistance reprogramming to MAPKi. METHODS: Flow cytometry was used to test TNFR1, TNFR2 and CD271 expression on, as well as NF-kB phosphorylation in human BRAF-mutant melanoma. The ability of melanoma cell lines to acquire MAPKi resistance in response to recombinant or macrophage-derived TNF was evaluated using the MTT cytotoxicity assay. Gene editing was implemented to knock out or knock in TNF receptors in melanoma cell lines. Knockout and knock-in cell line variants were employed to assess the intrinsic roles of these receptors in TNF-induced resistance to MAPKi. Multicolor immunofluorescence microscopy was utilized to test TNFR2 expression by melanoma in patients receiving MAPKi therapy. RESULTS: TNFR1 and TNFR2 are co-expressed at various levels on 4/7 BRAFV600E+ melanoma cell lines evaluated in this study. In vitro treatments with solTNF induce MAPKi resistance solely in TNFR2-expressing BRAFV600E+ melanoma cell lines. TNFR1 and TNFR2 knockout and knock-in studies indicate that solTNF-mediated MAPKi resistance in BRAFV600E+ melanomas is predicated on TNFR1 and TNFR2 co-expression, where TNFR1 is the central mediator of NF-kB signaling, while TNFR2 plays an auxiliary role. solTNF-mediated effects are transient and can be abrogated with biologics. Evaluation of patient specimens indicates that TNFR2 is expressed on 50% of primary BRAFV600E+ melanoma cells and that MAPKi therapy may lead to the enrichment of TNFR2-expressing tumor cells. CONCLUSIONS: Our data suggest that TNFR2 is essential to solTNF-induced MAPKi resistance and a possible biomarker to identify melanoma patients that can benefit from solTNF-targeting therapies.

Attenuated cell-cycle division protein 2 and elevated mitotic roles of polo-like kinase 1 characterize deficient myoblast fusion in peripheral arterial disease.

INTRODUCTION: We propose that MuSC-derived myoblasts in PAD have transcriptomic differences that can highlight underlying causes of ischemia-induced myopathy. METHODS: Differentiation capacity among perfused and ischemic human myoblasts was compared. Following next generation sequencing of mRNA, Ingenuity Pathway Analysis (IPA) was performed for canonical pathway enrichment. Live cell imaging and immunofluorescence were performed to determine myocyte fusion index and protein expression based on insights from IPA, specifically concerning cell cycle regulators including cell-division cycle protein 2 (CDC2) and polo-like kinase 1 (PLK1). RESULTS: Ischemic myoblasts formed attenuated myotubes indicative of reduced fusion. Additionally, myoblasts from ischemic segments showed significant differences in canonical pathways associated with PLK1 (upregulated) and G2/M DNA damage checkpoint regulation (downregulated). PLK1 inhibition with BI2536 did not affect cell viability in any group over 24 h but deterred fusion more significantly in PAD myoblasts. Furthermore, PLK1 inhibition reduced the expression of checkpoint protein CDC2 in perfused but not ischemic cells. CONCLUSION: Differentiating myoblasts derived from ischemic muscle have significant differences in gene expression including those essential to DNA-damage checkpoint regulation and cell cycle progress. DNA-damage checkpoint dysregulation may contribute to myopathy in PAD.

Inflammatory Caspase Activity Mediates HMGB1 Release and Differentiation in Myoblasts Affected by Peripheral Arterial Disease.

Introduction: We previously showed that caspase-1 and -11, which are activated by inflammasomes, mediate recovery from muscle ischemia in mice. We hypothesized that similar to murine models, inflammatory caspases modulate myogenicity and inflammation in ischemic muscle disease. Methods: Caspase activity was measured in ischemic and perfused human myoblasts in response to the NLRP3 and AIM2 inflammasome agonists (nigericin and poly(dA:dT), respectively) with and without specific caspase-1 or pan-caspase inhibition. mRNA levels of myogenic markers and caspase-1 were assessed, and protein levels of caspases-1, -4, -5, and -3 were measured by Western blot. Results: When compared to perfused cells, ischemic myoblasts demonstrated attenuated MyoD and myogenin and elevated caspase-1 mRNA. Ischemic myoblasts also had significantly higher enzymatic caspase activity with poly(dA:dT) (p < 0.001), but not nigericin stimulation. Inhibition of caspase activity including caspase-4/-5, but not caspase-1, blocked activation effects of poly(dA:dT). Ischemic myoblasts had elevated cleaved caspase-5. Inhibition of caspase activity deterred differentiation in ischemic but not perfused myoblasts and reduced the release of HMGB1 from both groups. Conclusion: Inflammatory caspases can be activated in ischemic myoblasts by AIM2 and influence ischemic myoblast differentiation and release of pro-angiogenic HMGB1. AIM2 inflammasome involvement suggests a role as a DNA damage sensor, and our data suggest that caspase-5 rather than caspase-1 may mediate the downstream mediator of this pathway.

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic ß-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.

Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity.

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

Most life processes require the energy produced by small cellular compartments called mitochondria. Many internal and external factors can harm these miniature powerhouses, potentially leading to cell death. For instance, in patients with Parkinson's or Alzheimer's disease, dying neurons often show mitochondrial damage. However, it is unclear exactly how injured mitochondria trigger the demise of these cells. Gaining a better understanding of this process requires studying the impact of mitochondrial damage in live neurons, something that is still difficult to do. As a response to this challenge, Xie, Jiao, Bai, Ilin et al. designed a new tool that can specifically injure mitochondria in the neurons of live zebrafish larvae at will, and fine-tune the amount of damage inflicted. The zebrafish are genetically engineered so that the mitochondria in their neurons carry a protein which can bind to a chemical compound called MG2I. When attached to each other, MG2I and the protein respond to far-red light by locally creating highly damaging chemicals. This means that whenever far-red light is shone onto the larvae, mitochondria in their neurons are harmed ­ the brighter the light, the stronger the damage. Zebrafish larvae exposed to these conditions immediately stopped swimming: mitochondria in their neurons could not produce enough energy and these cells could therefore no longer communicate properly. The neurons then started to die about 24 hours after exposure to the light, suggesting that the mitochondrial damage triggered other downstream processes that culminated in cell death. This new light-controlled tool could help to understand the consequences of mitochondrial damage, potentially revealing new ways to rescue impaired neurons in patients with Parkinson's or Alzheimer's disease. In the future, the method could be adapted to work in any type of cell and deactivate other cell compartments, so that it can be used to study many types of diseases.

An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex.

Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: the electron transfer chain (ETC), fatty acid ß-oxidation (FAO), and the tricarboxylic acid cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano-LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings provide a first view of an integrated molecular architecture for the major energy-generating pathways in mitochondria that ensures the safe transfer of unstable reducing equivalents from FAO to the ETC. They also offer insight into clinical ramifications for individuals with genetic defects in these pathways.

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission.

Many neurons influence their targets through co-release of neuropeptides and small-molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small-molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both stimulated emission depletion super-resolution and transmission electron microscopy. Ptp4E also controls the presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content.This article has an associated First Person interview with the first author of the paper.

Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor.

The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular and molecular processes including DNA damage detection and repair and transcription; indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjuncts given their capacity to impede genomic DNA repair in tumor cells. Conversely, overactivation of PARP can lead to NAD+ depletion, mitochondrial energy failure, and cell death. Since PARP activation facilitates genomic but impedes mitochondrial DNA repair, nonselective PARP inhibitors are likely to have opposing effects in these cellular compartments. Herein, we describe the synthesis and evaluation of the mitochondria-targeting PARP inhibitor, XJB-veliparib. Attachment of the hemigramicidin S pentapeptide isostere for mitochondrial targeting using a flexible linker at the primary amide site of veliparib did not disrupt PARP affinity or inhibition. XJB-veliparib was effective at low nanomolar concentrations (10-100 nM) and more potent than veliparib in protection from oxygen-glucose deprivation (OGD) in primary cortical neurons. Both XJB-veliparib and veliparib (10 nM) preserved mitochondrial NAD+ after OGD; however, only XJB-veliparib prevented release of NAD+ into cytosol. XJB-veliparib (10 nM) appeared to inhibit poly(ADP-ribose) polymer formation in mitochondria and preserve mitochondrial cytoarchitecture after OGD in primary cortical neurons. After 10 nM exposure, XJB-veliparib was detected by LC-MS in mitochondria but not nuclear-enriched fractions in neurons and was observed in mitoplasts stripped of the outer mitochondrial membrane obtained from HT22 cells. XJB-veliparib was also effective at preventing glutamate-induced HT22 cell death at micromolar concentrations. Importantly, in HT22 cells exposed to H2O2 to produce DNA damage, XJB-veliparib (10 µM) had no effect on nuclear DNA repair, in contrast to veliparib (10 µM) where DNA repair was retarded. XJB-veliparib and analogous mitochondria-targeting PARP inhibitors warrant further evaluation in vitro and in vivo, particularly in conditions where PARP overactivation leads to mitochondrial energy failure and maintenance of genomic DNA integrity is desirable, e.g., ischemia, oxidative stress, and radiation exposure.

Evidence for Compartmentalized Axonal Mitochondrial Biogenesis: Mitochondrial DNA Replication Increases in Distal Axons As an Early Response to Parkinson's Disease-Relevant Stress.

Dysregulation of mitochondrial biogenesis is implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). However, it is not clear how mitochondrial biogenesis is regulated in neurons, with their unique compartmentalized anatomy and energetic demands. This is particularly relevant in PD because selectively vulnerable neurons feature long, highly arborized axons where degeneration initiates. We previously found that exposure of neurons to chronic, sublethal doses of rotenone, a complex I inhibitor linked to PD, causes early increases in mitochondrial density specifically in distal axons, suggesting possible upregulation of mitochondrial biogenesis within axons. Here, we directly evaluated for evidence of mitochondrial biogenesis in distal axons and examined whether PD-relevant stress causes compartmentalized alterations. Using BrdU labeling and imaging to quantify replicating mitochondrial DNA (mtDNA) in primary rat neurons (pooled from both sexes), we provide evidence of mtDNA replication in axons along with cell bodies and proximal dendrites. We found that exposure to chronic, sublethal rotenone increases mtDNA replication first in neurites and later extending to cell bodies, complementing our mitochondrial density data. Further, isolating axons from cell bodies and dendrites, we discovered that rotenone exposure upregulates mtDNA replication in distal axons. Utilizing superresolution stimulated emission depletion (STED) imaging, we identified mtDNA replication at sites of mitochondrial-endoplasmic reticulum contacts in axons. Our evidence suggests that mitochondrial biogenesis occurs not only in cell bodies, but also in distal axons, and is altered under PD-relevant stress conditions in an anatomically compartmentalized manner. We hypothesize that this contributes to vulnerability in neurodegenerative diseases.SIGNIFICANCE STATEMENT Mitochondrial biogenesis is crucial for maintaining mitochondrial and cellular health and has been linked to neurodegenerative disease pathogenesis. However, regulation of this process is poorly understood in CNS neurons, which rely on mitochondrial function for survival. Our findings offer fundamental insight into these regulatory mechanisms by demonstrating that replication of mitochondrial DNA, an essential precursor for biogenesis, can occur in distal regions of CNS neuron axons independent of the soma. Further, this process is upregulated specifically in axons as an early response to neurodegeneration-relevant stress. This is the first demonstration of the compartmentalized regulation of CNS neuronal mitochondrial biogenesis in response to stress and may prove a useful target in development of therapeutic strategies for neurodegenerative disease.

4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses.

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to these therapies. The tumor microenvironment can impose metabolic restrictions on T cell function, creating a resistance mechanism to immunotherapy. We have previously shown tumor-infiltrating T cells succumb to progressive loss of metabolic sufficiency, characterized by repression of mitochondrial activity that cannot be rescued by PD-1 blockade. 4-1BB, a costimulatory molecule highly expressed on exhausted T cells, has been shown to influence metabolic function. We hypothesized that 4-1BB signaling might provide metabolic support to tumor-infiltrating T cells. 4-1BB costimulation of CD8+ T cells results in enhanced mitochondrial capacity (suggestive of fusion) and engages PGC1α-mediated pathways via activation of p38-MAPK. 4-1BB treatment of mice improves metabolic sufficiency in endogenous and adoptive therapeutic CD8+ T cells. 4-1BB stimulation combined with PD-1 blockade results in robust antitumor immunity. Sequenced studies revealed the metabolic support afforded by 4-1BB agonism need not be continuous and that a short course of anti-4-1BB pretreatment was sufficient to provide a synergistic response. Our studies highlight metabolic reprogramming as the dominant effect of 4-1BB therapy and suggest that combinatorial strategies using 4-1BB agonism may help overcome the immunosuppressive metabolic landscape of the tumor microenvironment.

Basics of Digital Microscopy.

Modern digital microscopy combines the equipment of classical light microscopy with a computerized imaging system. The technique comprises image formation by optics, image registration by a camera, and saving of image data in a computer file. This chapter describes limitations that are particular to each of these processes, including optical resolution, efficiency of image registration, characteristics of image file formats, and data management. Further suggestions are given which serve, in turn, to help construct a set of guidelines aimed at optimization of digital microscopic imaging. © 2018 by John Wiley & Sons, Inc.

α-Lipoic acid promotes α-tubulin hyperacetylation and blocks the turnover of mitochondria through mitophagy.

Lysine acetylation is tightly coupled to the nutritional status of the cell, as the availability of its cofactor, acetyl-CoA, fluctuates with changing metabolic conditions. Recent studies have demonstrated that acetyl-CoA levels act as an indicator of cellular nourishment, and increased abundance of this metabolite can block the induction of cellular recycling programmes. In the present study we investigated the cross-talk between mitochondrial metabolic pathways, acetylation and autophagy, using chemical inducers of mitochondrial acetyl-CoA production. Treatment of cells with α-lipoic acid (αLA), a cofactor of the pyruvate dehydrogenase complex, led to the unexpected hyperacetylation of α-tubulin in the cytosol. This acetylation was blocked by pharmacological inhibition of mitochondrial citrate export (a source for mitochondria-derived acetyl-CoA in the cytosol), was dependent on the α-tubulin acetyltransferase (αTAT) and was coupled to a loss in function of the cytosolic histone deacetylase, HDAC6. We further demonstrate that αLA slows the flux of substrates through autophagy-related pathways, and severely limits the ability of cells to remove depolarized mitochondria through PTEN-associated kinase 1 (PINK1)-mediated mitophagy.

Loss of Foxm1 Results in Reduced Somatotrope Cell Number during Mouse Embryogenesis.

FOXM1, a member of the forkhead box transcription factor family, plays a key role in cell cycling progression by regulating the expression of critical G1/S and G2/M phase transition genes. In vivo studies reveal that Foxm1 null mice have a 91% lethality rate at e18.5 due to significant cardiovascular and hepatic hypoplasia. Thus, FOXM1 has emerged as a key protein regulating mitotic division and cell proliferation necessary for embryogenesis. In the current study, we assess the requirement for Foxm1 in the developing pituitary gland. We find that Foxm1 is expressed in the pituitary at embryonic days 10.5-e18.5 and localizes with markers for active cell proliferation (BrdU). Interestingly, direct analysis of Foxm1 null mice at various embryonic ages, reveals no difference in gross pituitary morphology or cell proliferation. We do observe a downward trend in overall pituitary cell number and a small reduction in pituitary size in e18.5 embryos suggesting there may be subtle changes in pituitary proliferation not detected with our proliferation makers. Consistent with this, Foxm1 null mice have reductions in both the somatotrope and gonadotrope cell populations.

Impaired fertility and FSH synthesis in gonadotrope-specific Foxl2 knockout mice.

Impairments in pituitary FSH synthesis or action cause infertility. However, causes of FSH dysregulation are poorly described, in part because of our incomplete understanding of mechanisms controlling FSH synthesis. Previously, we discovered a critical role for forkhead protein L2 (FOXL2) in activin-stimulated FSH ß-subunit (Fshb) transcription in immortalized cells in vitro. Here, we tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, we selectively ablated Foxl2 in murine anterior pituitary gonadotrope cells. Conditional knockout (cKO) mice developed overtly normally but were subfertile in adulthood. Testis size and spermatogenesis were significantly impaired in cKO males. cKO females exhibited reduced ovarian weight and ovulated fewer oocytes in natural estrous cycles compared with controls. In contrast, ovaries of juvenile cKO females showed normal responses to exogenous gonadotropin stimulation. Both male and female cKO mice were FSH deficient, secondary to diminished pituitary Fshb mRNA production. Basal and activin-stimulated Fshb expression was similarly impaired in Foxl2 depleted primary pituitary cultures. Collectively, these data definitively establish FOXL2 as the first identified gonadotrope-restricted transcription factor required for selective FSH synthesis in vivo.