Chromosome-level organization of the regulatory genome in the Drosophila nervous system.

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.

Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo.

Paused RNA polymerase (Pol II) is a pervasive feature of Drosophila embryos and mammalian stem cells, but its role in development is uncertain. Here, we demonstrate that a spectrum of paused Pol II determines the "time to synchrony"-the time required to achieve coordinated gene expression across the cells of a tissue. To determine whether synchronous patterns of gene activation are significant in development, we manipulated the timing of snail expression, which controls the coordinated invagination of ∼1,000 mesoderm cells during gastrulation. Replacement of the strongly paused snail promoter with moderately paused or nonpaused promoters causes stochastic activation of snail expression and increased variability of mesoderm invagination. Computational modeling of the dorsal-ventral patterning network recapitulates these variable and bistable gastrulation profiles and emphasizes the importance of timing of gene activation in development. We conclude that paused Pol II and transcriptional synchrony are essential for coordinating cell behavior during morphogenesis.

Transcriptional coupling of distant regulatory genes in living embryos.

The prevailing view of metazoan gene regulation is that individual genes are independently regulated by their own dedicated sets of transcriptional enhancers. Past studies have reported long-range gene-gene associations1-3, but their functional importance in regulating transcription remains unclear. Here we used quantitative single-cell live imaging methods to provide a demonstration of co-dependent transcriptional dynamics of genes separated by large genomic distances in living Drosophila embryos. We find extensive physical and functional associations of distant paralogous genes, including co-regulation by shared enhancers and co-transcriptional initiation over distances of nearly 250 kilobases. Regulatory interconnectivity depends on promoter-proximal tethering elements, and perturbations in these elements uncouple transcription and alter the bursting dynamics of distant genes, suggesting a role of genome topology in the formation and stability of co-transcriptional hubs. Transcriptional coupling is detected throughout the fly genome and encompasses a broad spectrum of conserved developmental processes, suggesting a general strategy for long-range integration of gene activity.

The mouse cortico-basal ganglia-thalamic network.

The cortico-basal ganglia-thalamo-cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1-4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico-basal ganglia-thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

Cereblon influences the timing of muscle differentiation in Ciona tadpoles.

Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.

ERK signaling dissolves ERF repression condensates in living embryos.

Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor Ets-2 repressive factor (ERF), which has been linked to various human developmental disorders. Using high-resolution imaging, we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during interphase, providing visualization of a nuclear phase separation process regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.

Uncoupling neurogenic gene networks in the Drosophila embryo.

The EGF signaling pathway specifies neuronal identities in the Drosophila embryo by regulating developmental patterning genes such as intermediate neuroblasts defective (ind). EGFR is activated in the ventral midline and neurogenic ectoderm by the Spitz ligand, which is processed by the Rhomboid protease. CRISPR/Cas9 was used to delete defined rhomboid enhancers mediating expression at each site of Spitz processing. Surprisingly, the neurogenic ectoderm, not the ventral midline, was found to be the dominant source of EGF patterning activity. We suggest that Drosophila is undergoing an evolutionary transition in central nervous system (CNS)-organizing activity from the ventral midline to the neurogenic ectoderm.

Modeling transcriptional regulation of model species with deep learning.

To enable large-scale analyses of transcription regulation in model species, we developed DeepArk, a set of deep learning models of the cis-regulatory activities for four widely studied species: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, and Mus musculus DeepArk accurately predicts the presence of thousands of different context-specific regulatory features, including chromatin states, histone marks, and transcription factors. In vivo studies show that DeepArk can predict the regulatory impact of any genomic variant (including rare or not previously observed) and enables the regulatory annotation of understudied model species.

Transplanted human neural stem cells rescue phenotypes in zQ175 Huntington's disease mice and innervate the striatum.

Huntington's disease (HD), a genetic neurodegenerative disorder, primarily affects the striatum and cortex with progressive loss of medium-sized spiny neurons (MSNs) and pyramidal neurons, disrupting cortico-striatal circuitry. A promising regenerative therapeutic strategy of transplanting human neural stem cells (hNSCs) is challenged by the need for long-term functional integration. We previously described that, with short-term hNSC transplantation into the striatum of HD R6/2 mice, human cells differentiated into electrophysiologically active immature neurons, improving behavior and biochemical deficits. Here, we show that long-term (8 months) implantation of hNSCs into the striatum of HD zQ175 mice ameliorates behavioral deficits, increases brain-derived neurotrophic factor (BDNF) levels, and reduces mutant huntingtin (mHTT) accumulation. Patch clamp recordings, immunohistochemistry, single-nucleus RNA sequencing (RNA-seq), and electron microscopy demonstrate that hNSCs differentiate into diverse neuronal populations, including MSN- and interneuron-like cells, and form connections. Single-nucleus RNA-seq analysis also shows restoration of several mHTT-mediated transcriptional changes of endogenous striatal HD mouse cells. Remarkably, engrafted cells receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD.

Synaptic pathology in Huntington's disease: Beyond the corticostriatal pathway.

Huntington's disease (HD) is a heritable, fatal neurodegenerative disorder caused by a mutation in the Huntingtin gene. It is characterized by chorea, as well as cognitive and psychiatric symptoms. Histopathologically, there is a massive loss of striatal projection neurons and less but significant loss in other areas throughout the cortico-basal ganglia-thalamocortical (CBGTC) loop. The mutant huntingtin protein has been implicated in numerous functions, including an important role in synaptic transmission. Most studies on anatomical and physiological alterations in HD have focused on striatum and cerebral cortex. However, based on recent CBGTC projectome evidence, the need to study other pathways has become increasingly clear. In this review, we examine the current status of our knowledge of morphological and electrophysiological alterations of those pathways in animal models of HD. Based on recent studies, there is accumulating evidence that synaptic disconnection, particularly along excitatory pathways, is pervasive and almost universal in HD, thus supporting a critical role of the huntingtin protein in synaptic transmission.

Calcium dysregulation and compensation in cortical pyramidal neurons of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that predominantly affects striatal medium-sized spiny neurons and cortical pyramidal neurons (CPNs). It has been proposed that perturbations in Ca2+ homeostasis could play a role in CPN alterations. To test this hypothesis, we used the R6/2 mouse model of juvenile HD at different stages of disease progression; presymptomatic, early symptomatic, and late symptomatic. We combined whole-cell patch-clamp recordings of layer 2/3 CPNs with two-photon laser scanning microscopy to image somatic and dendritic Ca2+ transients associated with evoked action potentials (APs). We found that the amplitude of AP-induced Ca2+ transients recorded at the somata of CPNs was significantly reduced in presymptomatic and late symptomatic R6/2 mice compared with wild-type (WT) littermates. However, reduced amplitudes were compensated by increases in decay times, so that Ca2+ transient areas were similar between genotypes. AP-induced Ca2+ transients in CPN proximal dendrites were variable and differences did not reach statistical significance, except for reduced areas in the late symptomatic group. In late symptomatic mice, a specific store-operated Ca2+ channel antagonist, EVP4593, reduced somatic Ca2+ transient amplitude similarly in WT and R6/2 CPNs. In contrast, dantrolene, a ryanodine receptor (RyR) antagonist, and nifedipine, an L-type Ca2+ channel blocker, significantly reduced both somatic Ca2+ transient amplitude and area in R6/2 but not WT CPNs. These findings demonstrate that perturbations of Ca2+ homeostasis and compensation occur in CPNs before and after the onset of overt symptoms, and suggest RyRs and L-type Ca2+ channels as potential targets for therapeutic intervention.NEW & NOTEWORTHY We used two-photon microscopy to examine calcium influx induced by action potentials in cortical pyramidal neurons from a mouse model of Huntington's disease (HD), the R6/2. The amplitude of somatic calcium transients was reduced in R6/2 mice compared with controls. This reduction was compensated by increased decay times, which could lead to reduced calcium buffering capacity. L-type calcium channel and ryanodine receptor blockers reduced calcium transient area in HD neurons, suggesting new therapeutic avenues.

Early impairment of thalamocortical circuit activity and coherence in a mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive, fatal neurodegenerative disorder characterized by motor, cognitive, and psychiatric disturbances. There is no known cure for HD, but its progressive nature allows for early therapeutic intervention. Currently, much of the research has focused on the striatum, however, there is evidence suggesting that disruption of thalamocortical circuits could underlie some of the early symptoms of HD. Loss of both cortical pyramidal neurons (CPNs) and thalamic neurons occurs in HD patients, and cognitive, somatosensory, and attention deficits precede motor abnormalities. However, the role of thalamocortical pathways in HD progression has been understudied. Here, we measured single unit activity and local field potentials (LFPs) from electrode arrays implanted in the thalamus and primary motor cortex of 4-5 month-old male and female Q175 mice. We assessed neuronal activity under baseline conditions as well as during presentation of rewards delivered via actuation of an audible solenoid valve. HD mice showed a significantly delayed licking response to the reward stimulus. At the same time, neuronal activation to the reward was delayed in thalamic neurons, CPNs and fast-spiking cortical interneurons (FSIs) of HD mice. In addition, thalamocortical coherence increased at lower frequencies in HD relative to wildtype mice. Together, these data provide evidence that impaired cortical and thalamic responses to reward stimuli, and impaired thalamocortical coherence, may play an important early role in motor, cognitive, and learning deficits in HD patients.

Mutant huntingtin reduction in astrocytes slows disease progression in the BACHD conditional Huntington's disease mouse model.

Neuronal and non-neuronal cells express the huntingtin (HTT) protein, yet neurodegeneration in Huntington's disease (HD) is largely selective, affecting most prominently striatal medium spiny neurons and cortical pyramidal neurons. Selective toxicity of full-length human mutant HTT (fl-mHTT) may be due in part to its expression in non-neuronal cells. While studies suggest neuronal-glial interactions are important in HD and fl-mHTT is expressed in astrocytes, it has not been determined whether the expression of fl-mHTT in astrocytes is necessary for HD pathogenesis. To directly assess the necessity of fl-mHTT in astrocytes for HD pathogenesis, we used a mouse genetic approach and bred the conditional mHTT-expressing BACHD mouse model with GFAP-CreERT2 mice. We show that GFAP-CreERT2 expression in these mice is highly selective for astrocytes, and we are able to significantly reduce the expression of fl-mHTT protein in the striatum and cortex of BACHD/GFAP-CreERT2-tam mice. We performed behavioral, electrophysiological and neuropathological analyses of BACHD and BACHD/GFAP-CreERT2-tam mice. Behavioral analyses of BACHD/GFAP-CreERT2-tam mice demonstrate significant improvements in motor and psychiatric-like phenotypes. We observe improvements in neuropathological and electrophysiological phenotypes in BACHD/GFAP-CreERT2-tam mice compared to BACHD mice. We observed a restoration of the normal level αB-crystallin in the striatum of the BACHD/GFAP-CreERT2 mice, indicating a cell autonomous effect of mHTT on its expression. Taken together, this work indicates that astrocytes are important contributors to the progression of the behavioral and neuropathological phenotypes observed in HD.

Cortical Network Dynamics Is Altered in Mouse Models of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric disturbances. Although evidence indicates that projections from motor cortical areas play a key role in the development of dysfunctional striatal activity and motor phenotype, little is known about the changes in cortical microcircuits and their role in the development of the HD phenotype. Here we used two-photon laser-scanning microscopy to evaluate network dynamics of motor cortical neurons in layers II/III in behaving transgenic R6/2 and knock-in Q175+/- mice. Symptomatic R6/2 mice displayed increased motion manifested by a significantly greater number of motion epochs, whereas symptomatic Q175 mice displayed decreased motion. In both models, calcium transients in symptomatic mice displayed reduced amplitude, suggesting decreased bursting activity. Changes in frequency were genotype- and time-dependent; for R6/2 mice, the frequency was reduced during both motion and nonmotion, whereas in symptomatic Q175 mice, the reduction only occurred during nonmotion. In presymptomatic Q175 mice, frequency was increased during both behavioral states. Interneuronal correlation coefficients were generally decreased in both models, suggesting disrupted interneuronal communication in HD cerebral cortex. These results indicate similar and contrasting effects of the HD mutation on cortical ensemble activity depending on mouse model and disease stage.

Mechanisms underlying the enhancement of γ-aminobutyric acid responses in the external globus pallidus of R6/2 Huntington's disease model mice.

In Huntington's disease (HD), the output of striatal indirect pathway medium-sized spiny neurons (MSNs) is altered in its target region, the external globus pallidus (GPe). In a previous study we demonstrated that selective optogenetic stimulation of indirect pathway MSNs induced prolonged decay time of γ-aminobutyric acid (GABA) responses in GPe neurons. Here we identified the mechanism underlying this alteration. Electrophysiological recordings in slices from symptomatic R6/2 and wildtype (WT) mice were used to evaluate, primarily, the effects of GABA transporter (GAT) antagonists on responses evoked by optogenetic activation of indirect pathway MSNs. In addition, immunohistochemistry (IHC) and Western blots (WBs) were used to examine GAT-3 expression in HD and WT mice. A GAT-3 blocker (SNAP5114) increased decay time of GABA responses in WT and HD GPe neurons, but the effect was significantly greater in WT neurons. In contrast, a GAT-1 antagonist (NO-711) or a GABAB receptor antagonist (CGP 54626) produced small increases in decay time but no differential effects between genotypes. IHC and WBs showed reduction of GAT-3 expression in the GPe of HD mice. Thus, reduced expression or dysfunction of GAT-3 could underlie alterations of GPe responses to GABA inputs from striatum and could be a target for therapeutic intervention.

Striatal Direct and Indirect Pathway Output Structures Are Differentially Altered in Mouse Models of Huntington's Disease.

The present study examined synaptic communication between direct and indirect output pathway striatal medium-sized spiny neurons (MSNs) and their target structures, the substantia nigra pars reticulata (SNr) and the external globus pallidus (GPe) in two mouse models of Huntington's disease (HD). Cre recombination, optogenetics, and whole-cell patch-clamp recordings were used to determine alterations in intrinsic and synaptic properties of SNr and GPe neurons from both male and female symptomatic R6/2 (>60 d) and presymptomatic (2 months) or symptomatic (10-12 months) YAC128 mice. Cell membrane capacitance was decreased, whereas input resistance was increased in SNr neurons from R6/2, but not YAC128 mice. The amplitude of GABAergic responses evoked by optogenetic stimulation of direct pathway terminals was reduced in SNr neurons of symptomatic mice of both models. A decrease in spontaneous GABA synaptic activity, in particular large-amplitude events, in SNr neurons also was observed. Passive membrane properties of GPe neurons were not different between R6/2 or YAC128 mice and their control littermates. Similarly, the amplitude of GABA responses evoked by activation of indirect pathway MSN terminals and the frequency of spontaneous GABA synaptic activity were similar in HD and control animals. In contrast, the decay time of the evoked GABA response was significantly longer in cells from HD mice. Interestingly, activation of indirect pathway MSNs within the striatum evoked larger-amplitude responses in direct pathway MSNs. Together, these results demonstrate differential alterations in responses evoked by direct and indirect pathway terminals in SNr and GPe leading to striatal output imbalance and motor dysfunction.SIGNIFICANCE STATEMENT Previous work on Huntington's disease (HD) focused on striatal medium-sized spiny neurons (MSNs) almost exclusively. Little is known about the effects that alterations in the striatum have on output structures of the direct and indirect pathways, the substantia nigra pars reticulata (SNr) and the external segment of the globus pallidus (GPe), respectively. We combined electrophysiological and optogenetic methods to examine responses evoked by selective activation of terminals of direct and indirect pathway MSNs in SNr and GPe neurons in two mouse models of HD. We show a differential disruption of synaptic communication between the direct and indirect output pathways of the striatum with their target regions leading to an imbalance of striatal output, which will contribute to motor dysfunction.

Neural Deletion of Glucose Transporter Isoform 3 Creates Distinct Postnatal and Adult Neurobehavioral Phenotypes.

We created a neural-specific conditional murine glut3 (Slc2A3) deletion (glut3flox/flox/nestin-Cre+) to examine the effect of a lack of Glut3 on neurodevelopment. Compared with age-matched glut3flox/flox = WT and heterozygotes (glut3flox/+/nestin-Cre+), we found that a >90% reduction in male and female brain Glut3 occurred by postnatal day 15 (PN15) in glut3flox/flox/nestin-Cre+ This genetic manipulation caused a diminution in brain weight and cortical thickness at PN15, a reduced number of dendritic spines, and fewer ultrasonic vocalizations. Patch-clamp recordings of cortical pyramidal neurons revealed increased frequency of bicuculline-induced paroxysmal discharges as well as reduced latency, attesting to a functional synaptic and cortical hyperexcitability. Concomitant stunting with lower glucose concentrations despite increased milk intake shortened the lifespan, failing rescue by a ketogenic diet. This led to creating glut3flox/flox/CaMK2α-Cre+ mice lacking Glut3 in the adult male limbic system. These mice had normal lifespan, displayed reduced IPSCs in cortical pyramidal neurons, less anxiety/fear, and lowered spatial memory and motor abilities but heightened exploratory and social responses. These distinct postnatal and adult phenotypes, based upon whether glut3 gene is globally or restrictively absent, have implications for humans who carry copy number variations and present with neurodevelopmental disorders.SIGNIFICANCE STATEMENT Lack of the key brain-specific glucose transporter 3 gene found in neurons during early postnatal life results in significant stunting, a reduction in dendritic spines found on neuronal processes and brain size, heightened neuronal excitability, along with a shortened lifespan. When occurring in the adult and limited to the limbic system alone, lack of this gene in neurons reduces the fear of spatial exploration and socialization but does not affect the lifespan. These features are distinct heralding differences between postnatal and adult phenotypes based upon whether the same gene is globally or restrictively lacking. These findings have implications for humans who carry copy number variations pertinent to this gene and have been described to present with neurodevelopmental disorders.

Striatal GABAergic interneuron dysfunction in the Q175 mouse model of Huntington's disease.

The pathological hallmark of Huntington's disease (HD) is the massive loss of striatal and cortical neurons. Until recently, it was believed that striatal interneurons were spared from degeneration. This view has changed after the demonstration that parvalbumin (PV)-expressing interneurons also are vulnerable in humans. Here we compared morphological and functional changes of striatal fast-spiking interneurons (FSIs) and low-threshold spiking (LTS) interneurons in the Q175 mouse model of HD at presymptomatic (2 months) and symptomatic (12 months) stages of the disease. Electrophysiological intrinsic and synaptic properties of FSIs were significantly altered in symptomatic mice compared to wild-type (WT) littermates. Overall, FSIs became more excitable with disease progression. Sholl analysis also revealed a significant loss of dendritic complexity and excitatory synaptic inputs. The basic membrane and synaptic properties of LTS interneurons were similar in Q175 and WT mice regardless of disease stage. The resilience of LTS interneurons could be related to their sparsity of excitatory synaptic inputs compared with FSIs. However, in symptomatic mice, a subpopulation of LTS interneurons displayed an increase in action potential firing within oscillating bursts. Thus, we conclude that while both FSI and LTS interneurons demonstrate increases in excitability, the HD mutation differentially affects their membrane and synaptic properties as well as their ability to respond to compensatory challenges presented during the late stage of the disease. Alterations in GABAergic interneuron intrinsic activity and responsiveness to incoming signals may significantly affect SPN output thus contributing to abnormal motor movements in patients afflicted with HD.

Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers.

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.