Thyroid hormone remodels cortex to coordinate body-wide metabolism and exploration.

Animals adapt to environmental conditions by modifying the function of their internal organs, including the brain. To be adaptive, alterations in behavior must be coordinated with the functional state of organs throughout the body. Here, we find that thyroid hormone-a regulator of metabolism in many peripheral organs-directly activates cell-type-specific transcriptional programs in the frontal cortex of adult male mice. These programs are enriched for axon-guidance genes in glutamatergic projection neurons, synaptic regulatory genes in both astrocytes and neurons, and pro-myelination factors in oligodendrocytes, suggesting widespread plasticity of cortical circuits. Indeed, whole-cell electrophysiology revealed that thyroid hormone alters excitatory and inhibitory synaptic transmission, an effect that requires thyroid hormone-induced gene regulatory programs in presynaptic neurons. Furthermore, thyroid hormone action in the frontal cortex regulates innate exploratory behaviors and causally promotes exploratory decision-making. Thus, thyroid hormone acts directly on the cerebral cortex in males to coordinate exploratory behaviors with whole-body metabolic state.

Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.

Longevity, demographic characteristics, and socio-economic status are linked to triiodothyronine levels in the general population.

The hypothalamic-pituitary-thyroid (HPT) axis is fundamental to human biology, exerting central control over energy expenditure and body temperature. However, the consequences of normal physiologic HPT-axis variation in populations without diagnosed thyroid disease are poorly understood. Using nationally representative data from the 2007 to 2012 National Health and Nutrition Examination Survey, we explore relationships with demographic characteristics, longevity, and socio-economic factors. We find much larger variation across age in free T3 than other HPT-axis hormones. T3 and T4 have opposite relationships to mortality: free T3 is inversely related and free T4 is positively related to the likelihood of death. Free T3 and household income are negatively related, particularly at lower incomes. Finally, free T3 among older adults is associated with labor both in terms of unemployment and hours worked. Physiologic TSH/T4 explain only 1.7% of T3 variation, and neither are appreciably correlated to socio-economic outcomes. Taken together, our data suggest an unappreciated complexity of the HPT-axis signaling cascade broadly such that TSH and T4 may not be accurate surrogates of free T3. Furthermore, we find that subclinical variation in the HPT-axis effector hormone T3 is an important and overlooked factor linking socio-economic forces, human biology, and aging.

The CHARGE syndrome ortholog CHD-7 regulates TGF-ß pathways in Caenorhabditis elegans.

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant cellular pathways and targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, stress response, and body size determination. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the DAF-7/ tumor growth factor-ß (TGF-ß) pathway. Epistatic analysis places CHD-7 at the level of the DAF-3/DAF-5 complex, but we found that CHD-7 also directly impacts the expression of multiple components of this pathway. Transcriptomic analysis revealed that chd-7 mutants fail to repress daf-9 for execution of the dauer program. In addition, CHD-7 regulates the DBL-1/BMP pathway components and shares roles in male tail development and cuticle synthesis. To explore a potential conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino-mediated knockdown of Chd7 led to a reduction in col2a1 messenger RNA (mRNA) levels, a collagen whose expression depends on TGF-ß signaling. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by overexpression of col2a1 mRNA. We suggest that Chd7 has conserved roles in regulation of the TGF-ß signaling pathway and pathogenic Chd7 could lead to a defective extracellular matrix deposition.

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Publisher Correction: A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Flash memory: photochemical imprinting of neuronal action potentials onto a microbial rhodopsin.

We developed a technique, "flash memory", to record a photochemical imprint of the activity state--firing or not firing--of a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent states, D1 and D2, exist in a voltage-dependent equilibrium. A stable fluorescent state, F, is reached by a photochemical conversion from D2. When exposed to light of a wavelength λ(write), population transfers from D2 to F, at a rate determined by the D1 ⇌ D2 equilibrium. The population of F maintains a record of membrane voltage which persists in the dark. Illumination at a later time at a wavelength λ(read) excites fluorescence of F, probing this record. An optional third flash at a wavelength λ(reset) converts F back to D2, for a subsequent write-read cycle. The flash memory method offers the promise to decouple the recording of neural activity from its readout. In principle, the technique may enable one to generate snapshots of neural activity in a large volume of neural tissue, e.g., a complete mouse brain, by circumventing the challenge of imaging a large volume with simultaneous high spatial and high temporal resolution. The proof-of-principle flash memory sensors presented here will need improvements in sensitivity, speed, brightness, and membrane trafficking before this goal can be realized.

Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

DAF-12 regulates a connected network of genes to ensure robust developmental decisions.

The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that null daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of null daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post-dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions.

Transcriptional and spatiotemporal regulation of the dauer program.

Caenorhabditis elegans can enter a diapause stage called "dauer" when it senses that the environment is not suitable for development. This implies a detour from the typical developmental trajectory and requires a tight control of the developmental clock and a massive tissue remodeling. In the last decades, core components of the signaling pathways that govern the dauer development decision have been identified, but the tissues where they function for the acquisition of dauer-specific traits are still under intense study. Growing evidence demonstrates that these pathways engage in complex cross-talk and feedback loops. In this review, we summarize the current knowledge regarding the transcriptional regulation of the dauer program and the relevant tissues for its achievement. A better understanding of this process will provide insight on how developmental plasticity is achieved and how development decisions are under a robust regulation to ensure an all-or-nothing response. Furthermore, this developmental decision can also serve as a simplified model for relevant developmental disorders.Abbreviations: AID Auxin Induced Degron DA dafachronic acid Daf-c dauer formation constitutive Daf-d dauer formation defective DTC Distal Tip Cells ECM modified extracellular matrix GPCRs G protein-coupled receptors IIS insulin/IGF-1 signaling ILPs insulin-like peptides LBD Ligand Binding Domain PDL4 Post Dauer L4 TGF-ß transforming growth factor beta WT wild-type.

Sub-clinical triiodothyronine levels predict health, demographic, and socioeconomic outcomes.

The Hypothalamic-Pituitary-Thyroid (HPT) axis is fundamental to human biology, exerting central control over energy expenditure, metabolic rate, and body temperature. However, the consequences of "normal" physiologic HPT-axis variation in non-clinical populations are poorly understood. Using nationally-representative data from the 2007-2012 NHANES, we explore relationships with demographics, mortality, and socio-economic factors. We find much larger variation across age in free T3 than other HPT-axis hormones. T3 and T4 have opposite effects on mortality: free T3 is inversely related and free T4 is positively related with likelihood of death. Free T3 and household income are negatively related, particularly at lower incomes. Finally, free T3 among older adults is associated with labor both on the extensive margin (unemployment) and intensive margin (hours worked). Physiologic TSH/T4 explain only 1% of T3 variation, and neither are appreciably correlated to socio-economic outcomes. Taken together, our data suggest an unappreciated complexity and non-linearity of the HPT-axis signaling cascade broadly such that TSH and T4 may not be accurate surrogates of free T3. Furthermore, we find that sub-clinical variation in the HPT-axis effector hormone T3 is an important and overlooked factor linking socio-economic forces, human biology, and aging.

Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation.

The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) interacts with the Ldb1-Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1-Lhx1-Ssbp transcriptional complexes in embryonic development.

Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation.

The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem-affinity purification from kidney-induced Xenopus animal caps, we identified s ingle- s tranded DNA b inding p rotein 2 (Ssbp2) interacts with the Ldb1-Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1-Lhx1-Ssbp transcriptional complexes in embryonic development.

Thyroid hormone rewires cortical circuits to coordinate body-wide metabolism and exploratory drive.

Animals adapt to varying environmental conditions by modifying the function of their internal organs, including the brain. To be adaptive, alterations in behavior must be coordinated with the functional state of organs throughout the body. Here we find that thyroid hormone- a prominent regulator of metabolism in many peripheral organs- activates cell-type specific transcriptional programs in anterior regions of cortex of adult mice via direct activation of thyroid hormone receptors. These programs are enriched for axon-guidance genes in glutamatergic projection neurons, synaptic regulators across both astrocytes and neurons, and pro-myelination factors in oligodendrocytes, suggesting widespread remodeling of cortical circuits. Indeed, whole-cell electrophysiology recordings revealed that thyroid hormone induces local transcriptional programs that rewire cortical neural circuits via pre-synaptic mechanisms, resulting in increased excitatory drive with a concomitant sensitization of recruited inhibition. We find that thyroid hormone bidirectionally regulates innate exploratory behaviors and that the transcriptionally mediated circuit changes in anterior cortex causally promote exploratory decision-making. Thus, thyroid hormone acts directly on adult cerebral cortex to coordinate exploratory behaviors with whole-body metabolic state.

Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation.

cAMP is an ubiquitous second messenger. Localized areas with high cAMP concentration, i.e. cAMP microdomains, provide an elegant mechanism to generate signaling specificity and transduction efficiency. However, the mechanisms underlying cAMP effector targeting into these compartments is still unclear. Here we report the identification of radixin as a scaffolding unit for both cAMP effectors, Epac and PKA. This complex localizes in a submembrane compartment where cAMP synthesis occurs. Compartment disruption by shRNA and dominant negative approaches negatively affects cAMP action. Inhibition can be rescued by expression of Rap1b, a substrate for both Epac1 and PKA, but only in its GTP-bound and phosphorylated state. We propose that radixin scaffolds both cAMP effectors in a functional cAMP-sensing compartment for efficient signal transduction, using Rap1 as a downstream signal integrator.

Analysis of Thermogenesis Experiments with CalR.

Modern indirect calorimetry systems allow for high-frequency time series measurements of the factors affected by thermogenesis: energy intake and energy expenditure. These indirect calorimetry systems generate a flood of raw data recording oxygen consumption, carbon dioxide production, physical activity, and food intake among other factors. Analysis of these data requires time-consuming manual manipulation for formatting, data cleaning, quality control, and visualization. Beyond data handling, analyses of indirect calorimetry experiments require specialized statistical treatment to account for differential contributions of fat mass and lean mass to metabolic rates.Here we describe how to use the software package CalR version 1.2, to analyze indirect calorimetry data from three examples of thermogenesis, cold exposure, adrenergic agonism, and hyperthyroidism in mice, by providing standardized methods for reproducible research. CalR is a free online tool with an easy-to-use graphical user interface to import data files from the Columbus Instruments' CLAMS, Sable Systems' Promethion, and TSE Systems' PhenoMaster. Once loaded, CalR can quickly visualize experimental results and perform basic statistical analyses. We present a framework that standardizes the data structures and analyses of indirect calorimetry experiments to provide reusable and reproducible methods for the physiological data affecting body weight.

Activity-dependent regulome of human GABAergic neurons reveals new patterns of gene regulation and neurological disease heritability.

Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.

Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop.

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.

Anatomical and single-cell transcriptional profiling of the murine habenular complex.

The lateral habenula (LHb) is an epithalamic brain structure critical for processing and adapting to negative action outcomes. However, despite the importance of LHb to behavior and the clear anatomical and molecular diversity of LHb neurons, the neuron types of the habenula remain unknown. Here, we use high-throughput single-cell transcriptional profiling, monosynaptic retrograde tracing, and multiplexed FISH to characterize the cells of the mouse habenula. We find five subtypes of neurons in the medial habenula (MHb) that are organized into anatomical subregions. In the LHb, we describe four neuronal subtypes and show that they differentially target dopaminergic and GABAergic cells in the ventral tegmental area (VTA). These data provide a valuable resource for future study of habenular function and dysfunction and demonstrate neuronal subtype specificity in the LHb-VTA circuit.