Spatiotemporal modulation of growth factors directs the generation of multilineage mouse embryonic stem cell-derived mammary organoids.

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model.

ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones.

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.

Animal models for analysis of immunological responses to nanomaterials: Challenges and considerations.

Nanotechnology provides many solutions to improve conventional drug delivery and has a unique niche in the areas related to the specific targeting of the immune system, such as immunotherapies and vaccines. Preclinical studies in this field rely heavily on the combination of in vitro and in vivo methods to assess the safety and efficacy of nanotechnology platforms, nanoparticle-formulated drugs, and vaccines. While certain types of toxicities can be evaluated in vitro and good in vitro-in vivo correlation has been demonstrated for such tests, animal studies are still needed to address complex biological questions and, therefore, provide a unique contribution to establishing nanoparticle safety and efficacy profiles. The genetic, metabolic, mechanistic, and phenotypic diversity of currently available animal models often complicates both the animal choice and the interpretation of the results. This review summarizes current knowledge about differences in the immune system function and immunological responses of animals commonly used in preclinical studies of nanomaterials. We discuss challenges, highlight current gaps, and propose recommendations for animal model selection to streamline preclinical analysis of nanotechnology formulations.

Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote both cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive, although unrealized, therapeutic approach. T cells possess a unique mechanism of p38 mitogen-activated protein kinase (MAPK) activation downstream of T cell receptor (TCR) engagement through the phosphorylation of Tyr323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production. Here we show in human PDAC that a high percentage of infiltrating pY323(+) T cells was associated with large numbers of tumor necrosis factor (TNF)-α- and interleukin (IL)-17-producing CD4(+) tumor-infiltrating lymphocytes (TILs) and aggressive disease. The growth of mouse pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild-type CD4(+) T cells, but not those lacking the alternative pathway, enhanced tumor growth in T cell-deficient mice. Notably, a plasma membrane-permeable peptide derived from GADD45-α, the naturally occurring inhibitor of p38 pY323(+) (ref. 7), reduced CD4(+) TIL production of TNF-α, IL-17A, IL-10 and secondary cytokines, halted growth of implanted tumors and inhibited progression of spontaneous KRAS-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4(+) TILs results in alternative p38 activation and production of protumorigenic factors and can be targeted for therapeutic benefit.

Tissue specific loss of A-type lamins in the gastrointestinal epithelium can enhance polyp size.

The nuclear lamina, comprised of the A and B-type lamins, is important in maintaining nuclear shape and in regulating key nuclear functions such as chromatin organization and transcription. Deletion of the A-type lamins results in genome instability and many cancers show altered levels of A-type lamin expression. Loss of function mutations in the mouse Lmna gene result in early postnatal lethality, usually within 3-5 weeks of birth making an analysis of the role of lamins in carcinogenesis difficult. To circumvent early lethality, and determine the role of the A-type lamins in specific tissues in older mice we derived a conditional allele of Lmna(FL/FL) (floxed). Lmna(FL/FL) was specifically deleted in the gastrointestinal (GI) epithelium by crossing the Lmna(FL/FL) mice with Villin-Cre mice. Mice lacking Lmna in the GI are overtly normal with no effects on overall growth, longevity or GI morphology. On a GI specific sensitized (Apc(Min/+)) background, polyp numbers are unchanged, but polyp size is slightly increased, and only in the duodenum. Our findings reveal that although A-type lamins are dispensable in the postnatal GI epithelium, loss of Lmna under malignant conditions may, to a limited extent, enhance polyp size indicating that A-type lamins may regulate cell proliferation in the transformed GI epithelium.

Distinct cis regulatory elements govern the expression of TAG1 in embryonic sensory ganglia and spinal cord.

Cell fate commitment of spinal progenitor neurons is initiated by long-range, midline-derived, morphogens that regulate an array of transcription factors that, in turn, act sequentially or in parallel to control neuronal differentiation. Included among these are transcription factors that regulate the expression of receptors for guidance cues, thereby determining axonal trajectories. The Ig/FNIII superfamily molecules TAG1/Axonin1/CNTN2 (TAG1) and Neurofascin (Nfasc) are co-expressed in numerous neuronal cell types in the CNS and PNS - for example motor, DRG and interneurons - both promote neurite outgrowth and both are required for the architecture and function of nodes of Ranvier. The genes encoding TAG1 and Nfasc are adjacent in the genome, an arrangement which is evolutionarily conserved. To study the transcriptional network that governs TAG1 and Nfasc expression in spinal motor and commissural neurons, we set out to identify cis elements that regulate their expression. Two evolutionarily conserved DNA modules, one located between the Nfasc and TAG1 genes and the second directly 5' to the first exon and encompassing the first intron of TAG1, were identified that direct complementary expression to the CNS and PNS, respectively, of the embryonic hindbrain and spinal cord. Sequential deletions and point mutations of the CNS enhancer element revealed a 130bp element containing three conserved E-boxes required for motor neuron expression. In combination, these two elements appear to recapitulate a major part of the pattern of TAG1 expression in the embryonic nervous system.

Macrophage scavenger receptor A mediates the uptake of gold colloids by macrophages in vitro.

AIMS: While numerous studies have reported on nanoparticle uptake by phagocytic cells, the mechanisms of this uptake are poorly understood. A metastudy of research focusing on biological particulate matter has postulated that nanoparticles cannot be phagocytosed and therefore must enter cells via pinocytosis. The purpose of this study was to identify the route(s) of uptake of gold nanoparticles in vitro and to determine if these route(s) depend on particle size. MATERIALS & METHODS: The parent RAW264.7 cell line and its derivatives, transduced with a virus carrying siRNA to macrophage scavenger receptor A, were used as model phagocytes. Citrate-stabilized gold colloids were used as model nanoparticles. We used chemical inhibitors known to interfere with specific routes of particulate uptake. We developed multifocal light microscopy methods including multifocal stack analysis with NIH ImageJ software to analyze cell uptake. RESULTS: Irrespective of size, gold nanoparticles are internalized by macrophages via multiple routes, including both phagocytosis and pinocytosis. If either route was blocked, the particles entered cells via the other route. CONCLUSION: Gold nanoparticles with hydrodynamic sizes below 100 nm can be phagocytosed. Phagocytosis of anionic gold colloids by RAW264.7 cells is mediated by macrophage scavenger receptor A.

An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke.

Stroke is an age-related disease. Recovery after stroke is associated with axonal sprouting in cortex adjacent to the infarct. The molecular program that induces a mature cortical neuron to sprout a new connection after stroke is not known. We selectively isolated neurons that sprout a new connection in cortex after stroke and compared their whole-genome expression profile to that of adjacent, non-sprouting neurons. This 'sprouting transcriptome' identified a neuronal growth program that consists of growth factor, cell adhesion, axonal guidance and cytoskeletal modifying molecules that differed by age and time point. Gain and loss of function in three distinct functional classes showed new roles for these proteins in epigenetic regulation of axonal sprouting, growth factor-dependent survival of neurons and, in the aged mouse, paradoxical upregulation of myelin and ephrin receptors in sprouting neurons. This neuronal growth program may provide new therapeutic targets and suggest mechanisms for age-related differences in functional recovery.

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction.

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve-muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and ß-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

Development of a cell-based assay to quantify the inflammatory potential of test substances and screen compound libraries for anti-cancer drug candidates in a high-throughput format.

Despite the current availability of an impressive in vitro assay battery developed to quantitatively analyze the broad panel of small compounds and macromolecules that possess the inflammatory potential, little methodology exists nowadays that affords a researcher or clinician to quantify the ultimate output on the level of cell signaling response caused by inflammatory pathway stimulation. As a matter of fact, majority of analytical tools measure bona fide inflammatory substances (e.g., cytokines or chemokines) by their direct binding to secondary reagents such as specific antibodies or other selectively affine substrates with the final readout generated via quantification of the resulting complexes. Although specific and highly reproducible, this approach provides no discrimination between biologically active versus inactive input analyte nor does it address the differential biological potential for the questioned substances related to their in vivo stability and biodistribution. In a search for alternative solutions, a novel strategy is emerging that employs cell-based methods of inflammatory substance measurements allowing to detect and quantify the downstream effects of analyte's activity translated in terms of inflammatory pathways stimulation. In addition, application of cell based assays simultaneously permits entry level evaluation of compound toxicity and endows with a powerful approach to perform high-throughput screenings of, e.g., small molecule libraries in a quest for novel compounds capable of influencing the inflammation process.

At least ten genes define the imprinted Dlk1-Dio3 cluster on mouse chromosome 12qF1.

BACKGROUND: Genomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer. METHODOLOGY/PRINCIPAL FINDINGS: To identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression. CONCLUSIONS/SIGNIFICANCE: Ten imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/"Rian", AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.

The LEM domain proteins emerin and LAP2alpha are dispensable for human immunodeficiency virus type 1 and murine leukemia virus infections.

The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.

Neurotrypsin cleaves agrin locally at the synapse.

The synaptic serine protease neurotrypsin is considered to be essential for the establishment and maintenance of cognitive brain functions, because humans lacking functional neurotrypsin suffer from severe mental retardation. Neurotrypsin cleaves agrin at two homologous sites, liberating a 90-kDa and a C-terminal 22-kDa fragment from the N-terminal moiety of agrin. Morphological analyses indicate that neurotrypsin is contained in presynaptic terminals and externalized in association with synaptic activity, while agrin is localized to the extracellular space at or in the vicinity of the synapse. Here, we present a detailed biochemical analysis of neurotrypsin-mediated agrin cleavage in the murine brain. In brain homogenates, we found that neurotrypsin exclusively cleaves glycanated variants of agrin. Studies with isolated synaptosomes obtained by subcellular fractionation from brains of wild-type and neurotrypsin-overexpressing mice revealed that neurotrypsin-dependent cleavage of agrin was concentrated at synapses, where the most heavily glycanated variants of agrin predominate. Because agrin has been shown to play an important role in the formation and the maintenance of excitatory synapses in the central nervous system, its local cleavage at the synapse implicates the neurotrypsin/agrin system in the regulation of adaptive reorganizations of the synaptic circuitry in the context of cognitive functions, such as learning and memory.

The imprinted gene Magel2 regulates normal circadian output.

Mammalian circadian rhythms of activity are generated within the suprachiasmatic nucleus (SCN). Transcripts from the imprinted, paternally expressed Magel2 gene, which maps to the chromosomal region associated with Prader-Willi Syndrome (PWS), are highly enriched in the SCN. The Magel2 message is circadianly expressed and peaks during the subjective day. Mice deficient in Magel2 expression entrain to light cycles and express normal running-wheel rhythms, but with markedly reduced amplitude of activity and increased daytime activity. These changes are associated with reductions in food intake and male fertility. Orexin levels and orexin-positive neurons in the lateral hypothalamus are substantially reduced, suggesting that some of the consequences of Magel2 loss are mediated through changes in orexin signaling. The robust rhythmicity of Magel2 expression in the SCN and the altered behavioral rhythmicity of null mice reveal Magel2 to be a clock-controlled circadian output gene whose disruption results in some of the phenotypes characteristic of PWS.