Literature context: d A10042; RRIDs: AB_2534102 and AB_2534017; Invitrogen, Carlsbad, CA) befo
Ovarian development requires coordinate communications among oocytes, granulosa cells, and theca cells. Two Hedgehog (Hh) pathway ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), are produced by the granulosa cells and work together to regulate theca cell specification and development. Mice lacking both Dhh and Ihh had loss of normal ovarian function, which raised the question of which biological actions are specifically controlled by each ligand during folliculogenesis. By comparing the reproductive fitness, hormonal profiles, and ovarian transcriptomes among control, Dhh single-knockout (KO), Ihh KO, and Dhh/Ihh double-knockout (DKO) mice, we examined the specific roles of Dhh and Ihh in these processes. Dhh/Ihh DKO female mice were infertile because of a lack of theca cells and their steroid product androgen. Although Dhh and Ihh KO mice were fertile with normal folliculogenesis, they had decreased androgen production and alterations in their ovarian transcriptomes. Absence of Ihh led to aberrant steroidogenesis and elevated inflammation responses, which were not found in Dhh KO mouse ovaries, implicating that IHH has a greater impact than DHH on the activation of the Hh signaling pathway in the ovary. Our findings provide insight into not only how the Hh pathway influences folliculogenesis but also the distinct and overlapping roles of Dhh and Ihh in supporting ovarian development.
Literature context: Fisher Scientific Cat #A10042; RRID:AB_2534017 Donkey Anti-Goat IgG (H+L) High
Skeletal muscle cells (myofibers) are rod-shaped multinucleated cells surrounded by an extracellular matrix (ECM) basal lamina. In contrast to other cell types, nuclei in myofibers are positioned just below the plasma membrane at the cell periphery. Peripheral nuclear positioning occurs during myogenesis and is driven by myofibril crosslinking and contraction. Here we show that peripheral nuclear positioning is triggered by local accumulation of fibronectin secreted by myofibroblasts. We demonstrate that fibronectin via α5-integrin mediates peripheral nuclear positioning dependent on FAK and Src activation. Finally, we show that Cdc42, downstream of restricted fibronectin activation, is required for myofibril crosslinking but not myofibril contraction. Thus we identify that local activation of integrin by fibronectin secreted by myofibroblasts activates peripheral nuclear positioning in skeletal myofibers.
Literature context: ientific catalog number A10042, RRID:AB_2534017), donkey anti-mouse (1:500 Ther
Alterations in thalamic dopamine (DA) or DA D2 receptors (D2Rs) have been measured in drug addiction and schizophrenia, but the relevance of thalamic D2Rs for behavior is largely unknown. Using in situ hybridization and mice expressing green fluorescent protein (GFP) under the Drd2 promoter, we found that D2R expression within the thalamus is enriched in the paraventricular nucleus (PVT) as well as in more ventral midline thalamic nuclei. Within the PVT, D2Rs are inhibitory as their activation inhibits neuronal action potentials in brain slices. Using Cre-dependent anterograde and retrograde viral tracers, we further determined that PVT neurons are reciprocally interconnected with multiple areas of the limbic system including the amygdala and the nucleus accumbens (NAc). Based on these anatomical findings, we analyzed the role of D2Rs in the PVT in behaviors that are supported by these areas and that also have relevance for schizophrenia and drug addiction. Male and female mice with selective overexpression of D2Rs in the PVT showed attenuated cocaine locomotor sensitization, whereas anxiety levels, fear conditioning, sensorimotor gating, and food-motivated behaviors were not affected. These findings suggest the importance of PVT inhibition by D2Rs in modulating the sensitivity to cocaine, a finding that may have novel implications for human drug use.
Literature context: ation donkeyÎ±rabbit 568 A10042, RRID:AB_2534017) were applied in blocking buffe
The majority of neurons in the mammalian brain reside within the cerebellum (Cb). Yet, the evolutionary origins of the Cb are not well understood. There are several sensory nuclei present across vertebrate phylogeny collectively termed cerebellum-like structures (CbLS) due to a shared anatomy and physiology with the Cb. Despite the similarities, the CbLS are clearly not phylogenetically homologous with the Cb. Common structure and function may arise due to a shared genetic and developmental toolkit. To examine this possibility, we used sequence analysis, western blotting, immunohistochemistry and RT-qPCR to test for the expression of three genes that are critical for mammalian cerebellar development in the Cb and CbLS of an elasmobranch fish, Leucoraja erinacea. In the mammalian Cb, Pax6 is necessary for parallel fiber development, while Cbln1 and Grid2 code for proteins necessary for parallel fiber-principal cell synaptogenesis. Pax6 and Cbln1 are expressed by granule cells in the Cb and CbLS of the adult skate and stage 31 embryo. Grid2 is expressed by principal cells in the Cb and CbLS of the adult and stage 31 embryo. RT-qPCR showed this expression is spatially and temporally restricted to the Cb and CbLS. If Pax6, Cbln1 and Grid2 perform the same functions in the skate Cb and CbLS as they do in the mammalian Cb, then these structures may develop using a shared genetic toolkit and be considered generatively homologous. It is possible that the evolutionary genesis of the Cb was the result of duplication or expansion of the cerebellum-like developmental toolkit. This article is protected by copyright. All rights reserved.
Literature context: o Fisher Scientific Cat# A10042 RRID:AB_2534017, Lot #1476640
Action selection relies on the coordinated activity of striatal direct and indirect pathway medium spiny neurons (dMSNs and iMSNs, respectively). Loss of dopamine in Parkinson's disease is thought to disrupt this balance. While dopamine replacement with levodopa may restore normal function, the development of involuntary movements (levodopa-induced dyskinesia [LID]) limits therapy. How chronic dopamine loss and replacement with levodopa modulate the firing of identified MSNs in behaving animals is unknown. Using optogenetically labeled striatal single-unit recordings, we assess circuit dysfunction in parkinsonism and LID. Counter to current models, we found that following dopamine depletion, iMSN firing was elevated only during periods of immobility, while dMSN firing was dramatically and persistently reduced. Most notably, we identified a subpopulation of dMSNs with abnormally high levodopa-evoked firing rates, which correlated specifically with dyskinesia. These findings provide key insights into the circuit mechanisms underlying parkinsonism and LID, with implications for developing targeted therapies.
Literature context: bit Invitrogen Cat# A10042; RRID:AB_2534017 DAPI Sigma-Aldrich CAS: 28718-9
The ability to remember and to navigate to safe places is necessary for survival. Place navigation is known to involve medial entorhinal cortex (MEC)-hippocampal connections. However, learning-dependent changes in neuronal activity in the distinct circuits remain unknown. Here, by using optic fiber photometry in freely behaving mice, we discovered the experience-dependent induction of a persistent-task-associated (PTA) activity. This PTA activity critically depends on learned visual cues and builds up selectively in the MEC layer II-dentate gyrus, but not in the MEC layer III-CA1 pathway, and its optogenetic suppression disrupts navigation to the target location. The findings suggest that the visual system, the MEC layer II, and the dentate gyrus are essential hubs of a memory circuit for visually guided navigation.
Literature context: antibodies (Invitrogen, A10042 RRID:AB_2534017; A21202 RRID:AB_141607; A21447
Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders.
Literature context: Scientific Catalog No. A10042; RRID:AB_2534017 Chemicals, Peptides, and Recomb
The processing of triglyceride-rich lipoproteins (TRLs) in capillaries provides lipids for vital tissues, but our understanding of TRL metabolism is limited, in part because TRL processing and lipid movement have never been visualized. To investigate the movement of TRL-derived lipids in the heart, mice were given an injection of [2H]triglyceride-enriched TRLs, and the movement of 2H-labeled lipids across capillaries and into cardiomyocytes was examined by NanoSIMS. TRL processing and lipid movement in tissues were extremely rapid. Within 30 s, TRL-derived lipids appeared in the subendothelial spaces and in the lipid droplets and mitochondria of cardiomyocytes. Enrichment of 2H in capillary endothelial cells was not greater than in cardiomyocytes, implying that endothelial cells may not be a control point for lipid movement into cardiomyocytes. Remarkably, a deficiency of the putative fatty acid transport protein CD36, which is expressed highly in capillary endothelial cells, did not impede entry of TRL-derived lipids into cardiomyocytes.
Literature context: Life Technologies RRID:AB_2534017 Bacterial and Virus Strains
To interpret visual-motion events, the underlying computation must involve internal reference to the motion status of the observer's head. We show here that layer 6 (L6) principal neurons in mouse primary visual cortex (V1) receive a diffuse, vestibular-mediated synaptic input that signals the angular velocity of horizontal rotation. Behavioral and theoretical experiments indicate that these inputs, distributed over a network of 100 L6 neurons, provide both a reliable estimate and, therefore, physiological separation of head-velocity signals. During head rotation in the presence of visual stimuli, L6 neurons exhibit postsynaptic responses that approximate the arithmetic sum of the vestibular and visual-motion response. Functional input mapping reveals that these internal motion signals arrive into L6 via a direct projection from the retrosplenial cortex. We therefore propose that visual-motion processing in V1 L6 is multisensory and contextually dependent on the motion status of the animal's head.
Literature context: Fisher Scientific Cat# A10042, RRID:AB_2534017
MiR-122 is the most abundant miRNA in the human liver accounting for 52% of the entire hepatic miRNome. Previous studies have demonstrated that miR-122 is a valuable therapeutic target for liver diseases, including viral hepatitis, fibrosis, steatosis, and hepatocarcinoma. Here, we constructed a miR-122 doxycycline-inducible expression human embryonic stem cell line WAe001-A-15 using the piggyBac transposon system. The cell line retained its pluripotency, in vitro differentiation potential, normal morphology, and karyotype.
Literature context: 568 Thermo Fisher Cat#A10042; RRID:AB_2534017 donkey anti-rabbit; Alexa Fluor
Basal cells (BCs) are p63-expressing multipotent progenitors of skin, tracheoesophageal and urinary tracts. p63 is abundant in developing airways; however, it remains largely unclear how embryonic p63+ cells contribute to the developing and postnatal respiratory tract epithelium, and ultimately how they relate to adult BCs. Using lineage-tracing and functional approaches in vivo, we show that p63+ cells arising from the lung primordium are initially multipotent progenitors of airway and alveolar lineages but later become restricted proximally to generate the tracheal adult stem cell pool. In intrapulmonary airways, these cells are maintained immature to adulthood in bronchi, establishing a rare p63+Krt5- progenitor cell population that responds to H1N1 virus-induced severe injury. Intriguingly, this pool includes a CC10 lineage-labeled p63+Krt5- cell subpopulation required for a full H1N1-response. These data elucidate key aspects in the establishment of regionally distinct adult stem cell pools in the respiratory system, potentially with relevance to other organs.
Literature context: Thermo Fisher Scientific A10042 RRID:AB_2534017, 1:500), Alexa Fluor 488 Donkey
Astrocytes play an important role in Rett syndrome (RTT) disease progression. Although the non-cell-autonomous effect of RTT astrocytes on neurons was documented, cell-autonomous phenotypes and mechanisms within RTT astrocytes are not well understood. We report that spontaneous calcium activity is abnormal in RTT astrocytes in vitro, in situ, and in vivo. Such abnormal calcium activity is mediated by calcium overload in the endoplasmic reticulum caused by abnormal store operated calcium entry, which is in part dependent on elevated expression of TRPC4. Furthermore, the abnormal calcium activity leads to excessive activation of extrasynaptic NMDA receptors (eNMDARs) on neighboring neurons and increased network excitability in Mecp2 knockout mice. Finally, both the abnormal astrocytic calcium activity and the excessive activation of eNMDARs are caused by Mecp2 deletion in astrocytes in vivo. Our findings provide evidence that abnormal calcium homeostasis is a key cell-autonomous phenotype in RTT astrocytes, and reveal its mechanism and consequence.
Literature context: body (Life Technologies A10042, RRID:AB_2534017, 1:100) was then added to the c
The development of the peripheral nervous system relies on long-distance signaling from target organs back to the soma. In sympathetic neurons, this long-distance signaling is mediated by target derived Nerve Growth Factor (NGF) interacting with its axonal receptor, TrkA. This ligand receptor complex internalizes into what is commonly referred to as the signaling endosome which is transported retrogradely to the soma and dendrites to mediate survival signaling and synapse formation, respectively. The molecular identity of signaling endosomes in dendrites has not yet been determined. Here, we perform a detailed analysis of TrkA endosomal compartments and trafficking patterns. We find that signaling endosomes are not uniform but molecularly diversified into Rab7 (late endosome) and Rab11 (recycling endosome) populations in axons and dendrites in vitro and in the soma in vivo. Surprisingly, TrkA-NGF signaling endosomes in dendrites undergo dynamic trafficking events, including putative fusion and fission. Overall, we find that signaling endosomes do not remain as a singular endosomal subtype but instead exist in multiple populations that undergo dynamic endosomal trafficking events. These dynamic events might drive functional diversification of the signaling endosome.
Literature context: Fisher Scientific Cat# A10042, RRID:AB_2534017 Secondary antibodies Alexa Fluo
X-linked Adrenoleukodystrophy (X-ALD) is a genetic disease that caused by mutations in adenosine triphosphate [ATP]-binding-cassette transporter superfamily D member 1 (ABCD1) gene. We generated an induced pluripotent stem cell (iPSC) line from a 21-year-old male X-ALD patient-derived fibroblasts by Sendai virus mediated reprogramming. Established iPSCs stably expanded while maintaining immunoreactivity for various pluripotency markers and alkaline phosphatase, as well as normal 44+XY karyotype. Under the differentiation condition, the cells gave rise to cells of three germ layers.
Literature context: hermo Fisher Scientific A10042, RRID:AB_2534017 Donkey polyclonal anti-mouse Ig
Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.
Literature context: o Fisher Scientific Cat# A10042 RRID:AB_2534017 Anti-mouse IgG (H+L) Donkey Ale
Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain-sensing C-fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C-fibers: calcitonin-related polypeptide alpha (Calca/Cgrpα), specific to pain-sensing C-fibers, and tyrosine hydroxylase (Th), specific to low-threshold mechanoreceptive C-fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., ). Whole-mount cochlear preparations from 3-week- to 2-month-old CGRPα-EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II-like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co-expression could be observed, most abundantly in the middle cochlear turn.
Literature context: Fisher Scientific Cat# A10042; RRID:AB_2534017 Donkey anti-rat, Alexa Fluor 64
The differentiation of alveolar epithelial type I (AT1) and type II (AT2) cells is essential for the lung gas exchange function. Disruption of this process results in neonatal death or in severe lung diseases that last into adulthood. We developed live imaging techniques to characterize the mechanisms that control alveolar epithelial cell differentiation. We discovered that mechanical forces generated from the inhalation of amniotic fluid by fetal breathing movements are essential for AT1 cell differentiation. We found that a large subset of alveolar progenitor cells is able to protrude from the airway epithelium toward the mesenchyme in an FGF10/FGFR2 signaling-dependent manner. The cell protrusion process results in enrichment of myosin in the apical region of protruded cells; this myosin prevents these cells from being flattened by mechanical forces, thereby ensuring their AT2 cell fate. Our study demonstrates that mechanical forces and local growth factors synergistically control alveolar epithelial cell differentiation.
Literature context: (secondary antibody) Invitrogen RRID:AB_2534017
Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.
Literature context: gG (H+L) Invitrogen Cat#A10042; RRID:AB_2534017 Alexa Fluor 568 goat anti-guine
Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.
Literature context: Fisher Scientific Cat# A10042; RRID:AB_2534017 Donkey Anti-Rat IgG (H+L) Alexa
While the cell-intrinsic pathways governing beige adipocyte development and phenotype have been increasingly delineated, comparatively little is known about how beige adipocytes interact with other cell types in fat. Here, we introduce a whole-tissue clearing method for adipose that permits immunolabeling and three-dimensional profiling of structures including thermogenic adipocytes and sympathetic innervation. We found that tissue architecture and sympathetic innervation differ significantly between subcutaneous and visceral depots. Subcutaneous fat demonstrates prominent regional variation in beige fat biogenesis with localization of UCP1+ beige adipocytes to areas with dense sympathetic neurites. We present evidence that the density of sympathetic projections is dependent on PRDM16 in adipocytes, providing another potential mechanism underlying the metabolic benefits mediated by PRDM16. This powerful imaging tool highlights the interaction of tissue components during beige fat biogenesis and reveals a previously undescribed mode of regulation of the sympathetic nervous system by adipocytes.
Literature context: RRID:AB_2534017 Goat anti-Mouse Affinity Purifi
Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that histologically resembles embryonic skeletal muscle. RMS occurs throughout the body and an exclusively myogenic origin does not account for RMS occurring in sites devoid of skeletal muscle. We previously described an RMS model activating a conditional constitutively active Smoothened mutant (SmoM2) with aP2-Cre. Using genetic fate mapping, we show SmoM2 expression in Cre-expressing endothelial progenitors results in myogenic transdifferentiation and RMS. We show that endothelium and skeletal muscle within the head and neck arise from Kdr-expressing progenitors, and that hedgehog pathway activation results in aberrant expression of myogenic specification factors as a potential mechanism driving RMS genesis. These findings suggest that RMS can originate from aberrant development of non-myogenic cells.
Literature context: Fisher Scientific Cat# A10042, RRID:AB_2534017; Thermo Fisher Scientific Cat#
In the developing hypothalamus, the fat-derived hormone leptin stimulates the growth of axons from the arcuate nucleus of the hypothalamus (ARH) to other regions that control energy balance. These projections are significantly reduced in leptin deficient (Lepob/ob ) mice and this phenotype is largely rescued by neonatal leptin treatments. However, treatment of mature Lepob/ob mice is ineffective, suggesting that the trophic action of leptin is limited to a developmental critical period. To temporally delineate closure of this critical period for leptin-stimulated growth, we treated Lepob/ob mice with exogenous leptin during a variety of discrete time periods, and measured the density of Agouti-Related Peptide (AgRP) containing projections from the ARH to the ventral part of the dorsomedial nucleus of the hypothalamus (DMHv), and to the medial parvocellular part of the paraventricular nucleus (PVHmp). The results indicate that leptin loses its neurotrophic potential at or near postnatal day 28. The duration of leptin exposure appears to be important, with 9- or 11-day treatments found to be more effective than shorter (5-day) treatments. Furthermore, leptin treatment for 9 days or more was sufficient to restore AgRP innervation to both the PVHmp and DMHv in Lepob/ob females, but only to the DMHv in Lepob/ob males. Together, these findings reveal that the trophic actions of leptin are contingent upon timing and duration of leptin exposure, display both target and sex specificity, and that modulation of leptin-dependent circuit formation by each of these factors may carry enduring consequences for feeding behavior, metabolism, and obesity risk.
Literature context: at#A10042, A10037; RRID:AB_2534017; AB_2534013
Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.
Literature context: xa Fluor 568 Invitrogen A10042; RRID:AB_2534017 donkey anti-goat conjugated to
The mouse retina contains a single type of horizontal cell, a GABAergic interneuron that samples from all cone photoreceptors within reach and modulates their glutamatergic output via parallel feedback mechanisms. Because horizontal cells form an electrically coupled network, they have been implicated in global signal processing, such as large-scale contrast enhancement. Recently, it has been proposed that horizontal cells can also act locally at the level of individual cone photoreceptors. To test this possibility physiologically, we used two-photon microscopy to record light stimulus-evoked Ca2+ signals in cone axon terminals and horizontal cell dendrites as well as glutamate release in the outer plexiform layer. By selectively stimulating the two mouse cone opsins with green and UV light, we assessed whether signals from individual cones remain isolated within horizontal cell dendritic tips or whether they spread across the dendritic arbor. Consistent with the mouse's opsin expression gradient, we found that the Ca2+ signals recorded from dendrites of dorsal horizontal cells were dominated by M-opsin and those of ventral horizontal cells by S-opsin activation. The signals measured in neighboring horizontal cell dendritic tips varied markedly in their chromatic preference, arguing against global processing. Rather, our experimental data and results from biophysically realistic modeling support the idea that horizontal cells can process cone input locally, extending the classical view of horizontal cell function. Pharmacologically removing horizontal cells from the circuitry reduced the sensitivity of the cone signal to low frequencies, suggesting that local horizontal cell feedback shapes the temporal properties of cone output.
Literature context: Fisher Scientific Cat# A10042; RRID:AB_2534017 Rabbit Anti-Nanog Polyclonal An
Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.
Literature context: .thermofisher.com Cat # A10042; RRID:AB_2534017 Bacterial and Virus Strains
Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia.
Literature context: bit 568 [ThermoFisher, A-10042; RRID:AB_2534017], Donkey anti- mouse 488 [Therm
Detachment of newborn neurons from the neuroepithelium is required for correct neuronal architecture and functional circuitry. This process, also known as delamination, involves adherens-junction disassembly and acto-myosin-mediated abscission, during which the centrosome is retained while apical/ciliary membranes are shed. Cell-biological mechanisms mediating delamination are, however, poorly understood. Using live-tissue and super-resolution imaging, we uncover a centrosome-nucleated wheel-like microtubule configuration, aligned with the apical actin cable and adherens-junctions within chick and mouse neuroepithelial cells. These microtubules maintain adherens-junctions while actin maintains microtubules, adherens-junctions and apical end-foot dimensions. During neuronal delamination, acto-myosin constriction generates a tunnel-like actin-microtubule configuration through which the centrosome translocates. This movement requires inter-dependent actin and microtubule activity, and we identify drebrin as a potential coordinator of these cytoskeletal dynamics. Furthermore, centrosome compromise revealed that this organelle is required for delamination. These findings identify new cytoskeletal configurations and regulatory relationships that orchestrate neuronal delamination and may inform mechanisms underlying pathological epithelial cell detachment.
Literature context: Fisher Scientific Cat# A10042, RRID:AB_2534017), Alexa-647-conjugated donkey a
Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFPVglut2 , EYFPVgat , and GFPGad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.
Literature context: sÂ®, ThermoFisher, ScientificCat#A10042Alexa Fluorâ„¢ 647-conjugated phal
Many intracellular pathogens exploit host secretory trafficking to support their intracellular cycle, but knowledge of these pathogenic processes is limited. The bacterium Brucella abortus uses a type IV secretion system (VirB T4SS) to generate a replication-permissive Brucella-containing vacuole (rBCV) derived from the host ER, a process that requires host early secretory trafficking. Here we show that the VirB T4SS effector BspB contributes to rBCV biogenesis and Brucella replication by interacting with the conserved oligomeric Golgi (COG) tethering complex, a major coordinator of Golgi vesicular trafficking, thus remodeling Golgi membrane traffic and redirecting Golgi-derived vesicles to the BCV. Altogether, these findings demonstrate that Brucella modulates COG-dependent trafficking via delivery of a T4SS effector to promote rBCV biogenesis and intracellular proliferation, providing mechanistic insight into how bacterial exploitation of host secretory functions promotes pathogenesis.
Literature context: abbit IgG ThermoFisher RRID:AB_2534017 Alexa Fluor 647 donkey anti - m
The cortex is organized as a hierarchical processing structure. Feedback from higher levels of the hierarchy, known as top-down signals, have been shown to be involved in attentional and contextual modulation of sensory responses. Here we argue that top-down input to the primary visual cortex (V1) from A24b and the adjacent secondary motor cortex (M2) signals a prediction of visual flow based on motor output. A24b/M2 sends a dense and topographically organized projection to V1 that targets most neurons in layer 2/3. By imaging the activity of A24b/M2 axons in V1 of mice learning to navigate a 2D virtual environment, we found that their activity was strongly correlated with locomotion and resulting visual flow feedback in an experience-dependent manner. When mice were trained to navigate a left-right inverted virtual environment, correlations of neural activity with behavior reversed to match visual flow. These findings are consistent with a predictive coding interpretation of visual processing.
Literature context: o Fisher scientific Cat# A10042 RRID:AB_2534017 Chemicals, Peptides, and Recomb
The morphogenesis of branched organs remains a subject of abiding interest. Although much is known about the underlying signaling pathways, it remains unclear how macroscopic features of branched organs, including their size, network topology, and spatial patterning, are encoded. Here, we show that, in mouse mammary gland, kidney, and human prostate, these features can be explained quantitatively within a single unifying framework of branching and annihilating random walks. Based on quantitative analyses of large-scale organ reconstructions and proliferation kinetics measurements, we propose that morphogenesis follows from the proliferative activity of equipotent tips that stochastically branch and randomly explore their environment but compete neutrally for space, becoming proliferatively inactive when in proximity with neighboring ducts. These results show that complex branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple but generic rule, without recourse to a rigid and deterministic sequence of genetically programmed events.
Literature context: o. A10042 RRID:AB_2534017 Donkey ant
Purkinje cells of the primate cerebellum play critical but poorly understood roles in the execution of coordinated, accurate movements. Elucidating these roles has been hampered by a lack of techniques for manipulating spiking activity in these cells selectively-a problem common to most cell types in non-transgenic animals. To overcome this obstacle, we constructed AAV vectors carrying the channelrhodopsin-2 (ChR2) gene under the control of a 1 kb L7/Pcp2 promoter. We injected these vectors into the cerebellar cortex of rhesus macaques and tested vector efficacy in three ways. Immunohistochemical analyses confirmed selective ChR2 expression in Purkinje cells. Neurophysiological recordings confirmed robust optogenetic activation. Optical stimulation of the oculomotor vermis caused saccade dysmetria. Our results demonstrate the utility of AAV-L7-ChR2 for revealing the contributions of Purkinje cells to circuit function and behavior, and they attest to the feasibility of promoter-based, targeted, genetic manipulations in primates.
Literature context: bes Donkey; polyclonal 1:800 RRID:AB_2534017
Recent studies reveal complex patterns of hormone coexpression within enteroendocrine cells (EECs), contrary to the traditional view that gut hormones are expressed individually in EECs. Moreover, different hormones have been found in separate subcellular vesicles. However, detailed analysis of relative expression of multiple hormones has not been made. Subcellular studies have been confined to peptide hormones, and have not included the indolamine 5-hydroxytryptamine (5-HT) or the neuroendocrine protein chromogranin A (CgA). In the present work, coexpression of 5-HT, CgA, secretin, cholecystokinin (CCK), ghrelin, and glucagonlike peptide (GLP)-1 in mouse duodenum was quantified at a cellular and subcellular level by semiautomated cell counting and quantitative vesicle measurements. We investigated whether relative numbers of cells with colocalized hormones analyzed at a cell level matched the numbers revealed by examination of individual storage vesicles within cells. CgA and 5-HT were frequently expressed in EECs that contained combinations of GLP-1, ghrelin, secretin, and CCK. Separate subcellular stores of 5-HT, CgA, secretin, CCK, ghrelin, and GLP-1 were identified. In some cases, high-resolution analysis revealed small numbers of immunoreactive vesicles in cells dominated by a different hormone. Thus the observed incidence of cells with colocalized hormones is greater when analyzed at a subcellular, compared with a cellular, level. Subcellular analysis also showed that relative numbers of vesicles differ considerably between cells. Thus separate packaging of hormones that are colocalized is a general feature of EECs, and EECs exhibit substantial heterogeneity, including the colocalization of hormones that were formerly thought to be in cells of different lineages.
Literature context: Buenos Aires, Argentina), and a donkey anti-rabbit secondary antibody conjugated to Alexa 568 (Cat. # ab175470, 1Â :Â 500; Molecular Probes).For ON immunohistochemical stu
Glaucoma is a leading cause of blindness, characterized by retinal ganglion cell (RGC) loss and optic nerve (ON) damage. Cumulative evidence suggests glial cell involvement in the degeneration of the ON and RGCs. We analyzed the contribution of microglial reactivity to early axoglial alterations of the ON in an induced model of ocular hypertension. For this purpose, vehicle or chondroitin sulfate (CS) were weekly injected into the eye anterior chamber from Wistar rats for different intervals. The amount of Brn3a(+) RGC significantly decreased in CS-injected eyes for 10 and 15 (but not 6) weeks. A reduction in anterograde transport of β-subunit cholera toxin was observed in the superior colliculus and the lateral geniculate nucleus contralateral to CS-injected eyes for 6 and 15 weeks. A disruption of cholera toxin β-subunit transport was observed at the proximal myelinated ON. A significant decrease in phosphorylated neurofilament heavy chain immunoreactivity, an increase in ionized calcium-binding adaptor molecule 1(+), ED1(+) (microglial markers), and glial fibrillary acidic protein (astrocytes) (+) area, and decreased luxol fast blue staining were observed in the ON at 6 and 15 weeks of ocular hypertension. Microglial reactivity involvement was examined through a daily treatment with minocycline (30 mg/kg, i.p.) for 2 weeks, after 4 weeks of ocular hypertension. Minocycline prevented the increase in ionized calcium-binding adaptor molecule 1(+), ED-1(+), and glial fibrillary acidic protein(+) area, the decrease in phosphorylated neurofilament heavy-chain immunoreactivity and luxol fast blue staining, and the deficit in anterograde transport induced by 6 weeks of ocular hypertension. Thus, targeting microglial reactivity might prevent early axoglial alterations in the glaucomatous ON. Cover Image for this issue: doi: 10.1111/jnc.13807.
Literature context: : A10042, RRID:AB_2534017 Chemicals,
Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.
Literature context: t# A10042 RRID:AB_2534017), Donkey a
CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near -50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.
Literature context: # A10042; RRID:AB_2534017 IHC: Donke
The formation of functional synapses requires coordinated assembly of presynaptic transmitter release machinery and postsynaptic trafficking of functional receptors and scaffolds. Here, we demonstrate a critical role of presynaptic cadherin/catenin cell adhesion complexes in stabilizing functional synapses and spines in the developing neocortex. Importantly, presynaptic expression of stabilized β-catenin in either layer (L) 4 excitatory neurons or L2/3 pyramidal neurons significantly upregulated excitatory synaptic transmission and dendritic spine density in L2/3 pyramidal neurons, while its sparse postsynaptic expression in L2/3 neurons had no such effects. In addition, presynaptic β-catenin expression enhanced release probability of glutamatergic synapses. Newly identified β-catenin-interacting protein p140Cap is required in the presynaptic locus for mediating these effects. Together, our results demonstrate that cadherin/catenin complexes stabilize functional synapses and spines through anterograde signaling in the neocortex and provide important molecular evidence for a driving role of presynaptic components in spinogenesis in the neocortex.
Literature context: -A-10042; RRID:AB_2534017 Bacterial
Navigation, finding food sources, and avoiding danger critically depend on the identification and spatial localization of airborne chemicals. When monitoring the olfactory environment, rodents spontaneously engage in active olfactory sampling behavior, also referred to as exploratory sniffing . Exploratory sniffing is characterized by stereotypical high-frequency respiration, which is also reliably evoked by novel odorant stimuli [2, 3]. To study novelty-induced exploratory sniffing, we developed a novel, non-contact method for measuring respiration by infrared (IR) thermography in a behavioral paradigm in which novel and familiar stimuli are presented to head-restrained mice. We validated the method by simultaneously performing nasal pressure measurements, a commonly used invasive approach [2, 4], and confirmed highly reliable detection of inhalation onsets. We further discovered that mice actively orient their nostrils toward novel, previously unexperienced, smells. In line with the remarkable speed of olfactory processing reported previously [3, 5, 6], we find that mice initiate their response already within the first sniff after odor onset. Moreover, transecting the anterior commissure (AC) disrupted orienting, indicating that the orienting response requires interhemispheric transfer of information. This suggests that mice compare odorant information obtained from the two bilaterally symmetric nostrils to locate the source of the novel odorant. We further demonstrate that asymmetric activation of the anterior olfactory nucleus (AON) is both necessary and sufficient for eliciting orienting responses. These findings support the view that the AON plays an important role in the internostril difference comparison underlying rapid odor source localization.
Literature context: # A10042; RRID:AB_2534017; Molecular
ELOngation of Very Long chain fatty acids (ELOVL)-4 is essential for the synthesis of very long chain-fatty acids (fatty acids with chain lengths ≥ 28 carbons). The functions of ELOVL4 and its very long-chain fatty acid products are poorly understood at present. However, mutations in ELOVL4 cause neurodevelopmental or neurodegenerative diseases that vary according to the mutation and inheritance pattern. Heterozygous inheritance of different ELOVL4 mutations causes Stargardt-like Macular Dystrophy or Spinocerebellar Ataxia type 34. Homozygous inheritance of ELOVL4 mutations causes more severe disease characterized by seizures, intellectual disability, ichthyosis, and premature death. To better understand ELOVL4 and very long chain fatty acid function in the brain, we examined ELOVL4 expression in the mouse brain between embryonic day 18 and postnatal day 60 by immunolabeling using ELOVL4 and other marker antibodies. ELOVL4 was widely expressed in a region- and cell type-specific manner, and was restricted to cell bodies, consistent with its known localization to endoplasmic reticulum. ELOVL4 labeling was most prominent in gray matter, although labeling also was present in some cells located in white matter. ELOVL4 was widely expressed in the developing brain by embryonic day 18 and was especially pronounced in regions underlying the lateral ventricles and other neurogenic regions. The basal ganglia in particular showed intense ELOVL4 labeling at this stage. In the postnatal brain, cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, midbrain, pons, and medulla all showed prominent ELOVL4 labeling, although ELOVL4 distribution was not uniform across all cells or subnuclei within these regions. In contrast, the basal ganglia showed little ELOVL4 labeling in the postnatal brain. Double labeling studies showed that ELOVL4 was primarily expressed by neurons, although presumptive oligodendrocytes located in white matter tracts also showed labeling. Little or no ELOVL4 labeling was present in astrocytes or radial glial cells. These findings suggest that ELOVL4 and its very long chain fatty acid products are important in many parts of the brain and that they are particularly associated with neuronal function. Specific roles for ELOVL4 and its products in oligodendrocytes and myelin and in cellular proliferation, especially during development, are possible.
Literature context: i-Rabbit (RRID:AB_2534017, Thermo Fi
Organ formation is a multi-scale event that involves changes at the intracellular, cellular and tissue level. Organogenesis often starts with the formation of characteristically shaped organ precursors. However, the cellular mechanisms driving organ precursor formation are often not clear. Here, using zebrafish, we investigate the epithelial rearrangements responsible for the development of the hemispherical retinal neuroepithelium (RNE), a part of the optic cup. We show that in addition to basal shrinkage of RNE cells, active migration of connected epithelial cells into the RNE is a crucial player in its formation. This cellular movement is driven by progressive cell-matrix contacts and actively translocates prospective RNE cells to their correct location before they adopt neuroepithelial fate. Failure of this migration during neuroepithelium formation leads to ectopic determination of RNE cells and consequently impairs optic cup formation. Overall, this study illustrates how spatiotemporal coordination between morphogenic movements and fate determination critically influences organogenesis.
Literature context: #A-10042, RRID:AB_2534017 Donkey ant
Whether new neurons are added in the postnatal cerebral cortex is still debated. Here, we report that the meninges of perinatal mice contain a population of neurogenic progenitors formed during embryonic development that migrate to the caudal cortex and differentiate into Satb2+ neurons in cortical layers II-IV. The resulting neurons are electrically functional and integrated into local microcircuits. Single-cell RNA sequencing identified meningeal cells with distinct transcriptome signatures characteristic of (1) neurogenic radial glia-like cells (resembling neural stem cells in the SVZ), (2) neuronal cells, and (3) a cell type with an intermediate phenotype, possibly representing radial glia-like meningeal cells differentiating to neuronal cells. Thus, we have identified a pool of embryonically derived radial glia-like cells present in the meninges that migrate and differentiate into functional neurons in the neonatal cerebral cortex.
Literature context: 2 RRID:AB_2534017/Cat# A-315
The asymmetric location of stereociliary bundle (hair bundle) on the apical surface of mechanosensory hair cells (HCs) dictates the direction in which a given HC can respond to cues such as sound, head movements, and water pressure. Notably, vestibular sensory organs of the inner ear, the maculae, exhibit a line of polarity reversal (LPR) across which, hair bundles are polarized in a mirror-image pattern. Similarly, HCs in neuromasts of the zebrafish lateral line system are generated as pairs, and two sibling HCs develop opposite hair bundle orientations. Within these sensory organs, expression of the transcription factor Emx2 is restricted to only one side of the LPR in the maculae or one of the two sibling HCs in neuromasts. Emx2 mediates hair bundle polarity reversal in these restricted subsets of HCs and generates the mirror-image pattern of the sensory organs. Downstream effectors of Emx2 control bundle polarity cell-autonomously via heterotrimeric G proteins.
Literature context: # A10042; RRID:AB_2534017 Alexa 633
The thalamus receives sensory input from different circuits in the periphery. How these sensory channels are integrated at the level of single thalamic cells is not well understood. We performed targeted single-cell-initiated transsynaptic tracing to label the retinal ganglion cells that provide input to individual principal cells in the mouse lateral geniculate nucleus (LGN). We identified three modes of sensory integration by single LGN cells. In the first, 1-5 ganglion cells of mostly the same type converged from one eye, indicating a relay mode. In the second, 6-36 ganglion cells of different types converged from one eye, revealing a combination mode. In the third, up to 91 ganglion cells converged from both eyes, revealing a binocular combination mode in which functionally specialized ipsilateral inputs joined broadly distributed contralateral inputs. Thus, the LGN employs at least three modes of visual input integration, each exhibiting different degrees of specialization.
Literature context: # A10042; RRID:AB_2534017 Cy3-Strept
Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, we show that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca2+ influx through Dα7nAChRs or through LVA channels following GABAAR-mediated depolarizations. VIDEO ABSTRACT.
Literature context: # A10042, RRID:AB_2534017) against t
The formation of imprinted memories during a critical period is crucial for vital behaviors, including filial attachment. Yet, little is known about the underlying molecular mechanisms. Using a combination of behavior, pharmacology, in vivo surface sensing of translation (SUnSET) and DiOlistic labeling we found that, translational control by the eukaryotic translation initiation factor 2 alpha (eIF2α) bidirectionally regulates auditory but not visual imprinting and related changes in structural plasticity in chickens. Increasing phosphorylation of eIF2α (p-eIF2α) reduces translation rates and spine plasticity, and selectively impairs auditory imprinting. By contrast, inhibition of an eIF2α kinase or blocking the translational program controlled by p-eIF2α enhances auditory imprinting. Importantly, these manipulations are able to reopen the critical period. Thus, we have identified a translational control mechanism that selectively underlies auditory imprinting. Restoring translational control of eIF2α holds the promise to rejuvenate adult brain plasticity and restore learning and memory in a variety of cognitive disorders.
Bone marrow (BM)-derived cells (BMDCs) contribute to endometrial regeneration. Our objective was to develop a nongonadotoxic mouse BM transplant (BMT) model using 5-fluorouracil (5-FU) for investigating BMDCs trafficking in reproduction. Female C57BL/6J mice received either single (CTX-1) or paired (CTX-2) 5-FU (150 mg/kg) dose, or single (CTX-1+SCF) or paired-dose (CTX-3+SCF) 5-FU with stem cell factor (SCF). Control mice received BMT only or saline. BM cells (20 × 106) from transgenic green-fluorescent protein (GFP) mice were injected iv. For fertility experiment, mice were mated on day 28 after BMT. Alternatively, mice were killed 1 month after BMT and BMDCs recruitment to the uterus was determined. Mice receiving 5-FU ± SCF showed intact ovarian function and fertility. CTX-3+SCF resulted in greatest BM donor chimerism at 1 month (∼45%). Flow cytometry analysis demonstrated that 6.6% of total uterine cells in CTX-3+SCF mice were GFP+ BMDCs. Remarkably, this was about 40- and 80-fold greater than BMDCs in uterus of CTX-1 or BMT only mice (6.6% vs 0.16% vs 0.08%, respectively, P < .001). Immunohistochemical analysis showed that BMDCs in the uterus were mostly localized to the endometrial stroma (71.8%). The majority of endometrial BMDCs colocalized with the pan-leuokocyte CD45 marker (58.5%), but 41.5% were CD45-negative. Cytokeratin and CD31 staining showed that the GFP+CD45- cells were not epithelial or endothelial, confirming their stromal identity. We demonstrate that paired-dose 5-FU regimen results in efficient BM donor chimerism while maintaining ovarian function and fertility. This model could be used for studying BMDCs trafficking to the uterus in various reproductive physiological and pathological conditions.
Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.
Relaxin-like factor, commonly known as insulin-like factor (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. The study aimed to identify a relaxin family peptide receptor 2 (RXFP2)-specific antibody suitable for immunological approaches, analyze which testicular germ cell types express RXFP2, and clarify its expression dynamics in the boar testis. In addition, the function of INSL3-RXFP2 signaling on the germ cells was explored by neutralizing INSL3 using long-term active immunization. Samples were collected from Duroc boars, and a commercially available RXFP2-specific antibody directed against the human RXFP2 endodomain was identified by characterizing its specificity in HEK-293 cells expressing mouse RXFP2, and by demonstrating the suitability for analyzing RXFP2 expression in porcine tissues. RXFP2 mRNA and protein were both localized mainly in meiotic and post-meiotic germ cells, but not in Leydig cells. Functional RXFP2, which enables INSL3 to bind, was detected as an ∼85-kDa band, which increased in intensity from the pubertal stage onward. Interestingly, INSL3 immunization significantly reduced testis weight and induced a 4-fold increase in the frequency of apoptotic germ cells, which was associated with the up-regulation of pro-apoptotic caspase-3 (CASP3) and BAX, and the down-regulation of anti-apoptotic XIAP and BCL2, and a substantial reduction in sperm concentration. These results revealed that RXFP2 was expressed in boar meiotic and post-meiotic germ cells, where INSL3 neutralization led to increased germ cell apoptosis and reduced sperm output, suggesting that INSL3 acts as a survival/anti-apoptotic factor in maintaining sperm production.