Advances in transposable elements: from mechanisms to applications in mammalian genomics.

It has been 70 years since Barbara McClintock discovered transposable elements (TE), and the mechanistic studies and functional applications of transposable elements have been at the forefront of life science research. As an essential part of the genome, TEs have been discovered in most species of prokaryotes and eukaryotes, and the relative proportion of the total genetic sequence they comprise gradually increases with the expansion of the genome. In humans, TEs account for about 40% of the genome and are deeply involved in gene regulation, chromosome structure maintenance, inflammatory response, and the etiology of genetic and non-genetic diseases. In-depth functional studies of TEs in mammalian cells and the human body have led to a greater understanding of these fundamental biological processes. At the same time, as a potent mutagen and efficient genome editing tool, TEs have been transformed into biological tools critical for developing new techniques. By controlling the random insertion of TEs into the genome to change the phenotype in cells and model organisms, critical proteins of many diseases have been systematically identified. Exploiting the TE's highly efficient in vitro insertion activity has driven the development of cutting-edge sequencing technologies. Recently, a new technology combining CRISPR with TEs was reported, which provides a novel targeted insertion system to both academia and industry. We suggest that interrogating biological processes that generally depend on the actions of TEs with TEs-derived genetic tools is a very efficient strategy. For example, excessive activation of TEs is an essential factor in the occurrence of cancer in humans. As potent mutagens, TEs have also been used to unravel the key regulatory elements and mechanisms of carcinogenesis. Through this review, we aim to effectively combine the traditional views of TEs with recent research progress, systematically link the mechanistic discoveries of TEs with the technological developments of TE-based tools, and provide a comprehensive approach and understanding for researchers in different fields.

Variable task switching in the feeding network of Aplysia is a function of differential command input.

Command systems integrate sensory information and then activate the interneurons and motor neurons that mediate behavior. Much research has established that the higher-order projection neurons that constitute these systems can play a key role in specifying the nature of the motor activity induced, or determining its parametric features. To a large extent, these insights have been obtained by contrasting activity induced by stimulating one neuron (or set of neurons) to activity induced by stimulating a different neuron (or set of neurons). The focus of our work differs. We study one type of motor program, ingestive feeding in the mollusc Aplysia californica, which can either be triggered when a single projection neuron (CBI-2) is repeatedly stimulated or can be triggered by projection neuron coactivation (e.g., activation of CBI-2 and CBI-3). We ask why this might be an advantageous arrangement. The cellular/molecular mechanisms that configure motor activity are different in the two situations because the released neurotransmitters differ. We focus on an important consequence of this arrangement, the fact that a persistent state can be induced with repeated CBI-2 stimulation that is not necessarily induced by CBI-2/3 coactivation. We show that this difference can have consequences for the ability of the system to switch from one type of activity to another.NEW & NOTEWORTHY We study a type of motor program that can be induced either by stimulating a higher-order projection neuron that induces a persistent state, or by coactivating projection neurons that configure activity but do not produce a state change. We show that when an activity is configured without a state change, it is possible to immediately return to an intermediate state that subsequently can be converted to any type of motor program.

An inter-organ neural circuit for appetite suppression.

Glucagon-like peptide-1 (GLP-1) is a signal peptide released from enteroendocrine cells of the lower intestine. GLP-1 exerts anorectic and antimotility actions that protect the body against nutrient malabsorption. However, little is known about how intestinal GLP-1 affects distant organs despite rapid enzymatic inactivation. We show that intestinal GLP-1 inhibits gastric emptying and eating via intestinofugal neurons, a subclass of myenteric neurons that project to abdominal sympathetic ganglia. Remarkably, cell-specific ablation of intestinofugal neurons eliminated intestinal GLP-1 effects, and their chemical activation functioned as a GLP-1 mimetic. GLP-1 sensing by intestinofugal neurons then engaged a sympatho-gastro-spinal-reticular-hypothalamic pathway that links abnormal stomach distension to craniofacial programs for food rejection. Within this pathway, cell-specific activation of discrete neuronal populations caused systemic GLP-1-like effects. These molecularly identified, delimited enteric circuits may be targeted to ameliorate the abdominal bloating and loss of appetite typical of gastric motility disorders.

The Complement of Projection Neurons Activated Determines the Type of Feeding Motor Program in Aplysia.

Multiple projection neurons are often activated to initiate behavior. A question that then arises is, what is the unique functional role of each neuron activated? We address this issue in the feeding system of Aplysia. Previous experiments identified a projection neuron [cerebral buccal interneuron 2 (CBI-2)] that can trigger ingestive motor programs but only after it is repeatedly stimulated, i.e., initial programs are poorly defined. As CBI-2 stimulation continues, programs become progressively more ingestive (repetition priming occurs). This priming results, at least in part, from persistent actions of peptide cotransmitters released from CBI-2. We now show that in some preparations repetition priming does not occur. There is no clear seasonal effect; priming and non-priming preparations are encountered throughout the year. CBI-2 is electrically coupled to a second projection neuron, cerebral buccal interneuron 3 (CBI-3). In preparations in which priming does not occur, we show that ingestive activity is generated when CBI-2 and CBI-3 are coactivated. Programs are immediately ingestive, i.e., priming is not necessary, and a persistent state is not induced. Our data suggest that dynamic changes in the configuration of activity can vary and be determined by the complement of projection neurons that trigger activity.

Persistent effects of cyclic adenosine monophosphate are directly responsible for maintaining a neural network state.

Network states are often determined by modulators that alter the synaptic and cellular properties of the constituent neurons. Frequently neuromodulators act via second messengers, consequently their effects can persist. This persistence at the cellular/molecular level determines the maintenance of the state at the network level. Here we study a feeding network in Aplysia. In this network, persistent modulation supports the maintenance of an ingestive state, biasing the network to generate ingestive motor programs. Neuropeptides that exert cyclic adenosine monophosphate (cAMP) dependent effects play an important role in inducing the ingestive state. Most commonly, modulatory effects exerted through cAMP signaling are persistent as a consequence of PKA activation. This is not the case in the neurons we study. Instead maintenance of the network state depends on the persistence of cAMP itself. Data strongly suggest that this is a consequence of the direct activation of a cyclic nucleotide gated current.

A Neural Circuit for Gut-Induced Reward.

The gut is now recognized as a major regulator of motivational and emotional states. However, the relevant gut-brain neuronal circuitry remains unknown. We show that optical activation of gut-innervating vagal sensory neurons recapitulates the hallmark effects of stimulating brain reward neurons. Specifically, right, but not left, vagal sensory ganglion activation sustained self-stimulation behavior, conditioned both flavor and place preferences, and induced dopamine release from Substantia nigra. Cell-specific transneuronal tracing revealed asymmetric ascending pathways of vagal origin throughout the CNS. In particular, transneuronal labeling identified the glutamatergic neurons of the dorsolateral parabrachial region as the obligatory relay linking the right vagal sensory ganglion to dopamine cells in Substantia nigra. Consistently, optical activation of parabrachio-nigral projections replicated the rewarding effects of right vagus excitation. Our findings establish the vagal gut-to-brain axis as an integral component of the neuronal reward pathway. They also suggest novel vagal stimulation approaches to affective disorders.

Cellular Effects of Repetition Priming in the Aplysia Feeding Network Are Suppressed during a Task-Switch But Persist and Facilitate a Return to the Primed State.

Many neural networks are multitasking and receive modulatory input, which configures activity. As a result, these networks can enter a relatively persistent state in which they are biased to generate one type of output as opposed to another. A question we address is as follows: what happens to this type of state when the network is forced to task-switch? We address this question in the feeding system of the mollusc Aplysia This network generates ingestive and egestive motor programs. We focus on an identified neuron that is selectively active when programs are ingestive. Previous work has established that the increase in firing frequency observed during ingestive programs is at least partially mediated by an excitability increase. Here we identify the underlying cellular mechanism as the induction of a cAMP-dependent inward current. We ask how this current is impacted by the subsequent induction of egestive activity. Interestingly, we demonstrate that this task-switch does not eliminate the inward current but instead activates an outward current. The induction of the outward current obviously reduces the net inward current in the cell. This produces the decrease in excitability and firing frequency required for the task-switch. Importantly, however, the persistence of the inward current is not impacted. It remains present and coexists with the outward current. Consequently, when effects of egestive priming and the outward current dissipate, firing frequency and excitability remain above baseline levels. This presumably has important functional implications in that it will facilitate a return to ingestive activity.SIGNIFICANCE STATEMENT Under physiological conditions, an animal generating a particular type of motor activity can be forced to at least briefly task-switch. In some circumstances, this involves the temporary induction of an "antagonistic" or incompatible motor program. For example, ingestion can be interrupted by a brief period of egestive activity. In this type of situation, it is often desirable for behavioral switching to occur rapidly and efficiently. In this report, we focus on a particular aspect of this type of task-switch. We determine how the priming that occurs when a multitasking network repeatedly generates one type of motor activity can be retained during the execution of an incompatible motor program.

Use of the Aplysia feeding network to study repetition priming of an episodic behavior.

Many central pattern generator (CPG)-mediated behaviors are episodic, meaning that they are not continuously ongoing; instead, there are pauses between bouts of activity. This raises an interesting possibility, that the neural networks that mediate these behaviors are not operating under "steady-state" conditions; i.e., there could be dynamic changes in motor activity as it stops and starts. Research in the feeding system of the mollusk Aplysia californica has demonstrated that this can be the case. After a pause, initial food grasping responses are relatively weak. With repetition, however, responses strengthen. In this review we describe experiments that have characterized cellular/molecular mechanisms that produce these changes in motor activity. In particular, we focus on cumulative effects of modulatory neuropeptides. Furthermore, we relate Aplysia research to work in other systems and species, and develop a hypothesis that postulates that changes in response magnitude are a reflection of an efficient feeding strategy.

Neuromodulation as a mechanism for the induction of repetition priming.

It is becoming apparent that the activity of many neural networks is shaped by effects of endogenous neuromodulators. Modulators exert second messenger-mediated actions that persist. We consider how this may impact network function and its potential role in the induction of repetition priming (increased performance when behavior is repeated). When effects of modulators persist and modulatory substances are repeatedly released, their effects will accumulate (summate) and become more pronounced. If this enhances the ability of a network to generate a particular output, performance will improve. We review data that support this model, and consider its implications for task switching. This model predicts that priming of one type of network activity will negatively impact the rapid transition to an incompatible type.

Complementary interactions between command-like interneurons that function to activate and specify motor programs.

Motor activity is often initiated by a population of command-like interneurons. Command-like interneurons that reliably drive programs have received the most attention, so little is known about how less reliable command-like interneurons may contribute to program generation. We study two electrically coupled interneurons, cerebral-buccal interneuron-2 (CBI-2) and CBI-11, which activate feeding motor programs in the mollusk Aplysia californica. Earlier work indicated that, in rested preparations, CBI-2, a powerful activator of programs, can trigger ingestive and egestive programs. CBI-2 reliably generated ingestive patterns only when it was repeatedly stimulated. The ability of CBI-2 to trigger motor activity has been attributed to the two program-promoting peptides it contains, FCAP and CP2. Here, we show that CBI-11 differs from CBI-2 in that it contains FCAP but not CP2. Furthermore, it is weak in its ability to drive programs. On its own, CBI-11 is therefore less effective as a program activator. When it is successful, however, CBI-11 is an effective specifier of motor activity; that is, it drives mostly ingestive programs. Importantly, we found that CBI-2 and CBI-11 complement each other's actions. First, prestimulation of CBI-2 enhanced the ability of CBI-11 to drive programs. This effect appears to be partly mediated by CP2. Second, coactivation of CBI-11 with CBI-2 makes CBI-2 programs immediately ingestive. This effect may be mediated by specific actions that CBI-11 exerts on pattern-generating interneurons. Therefore, different classes of command-like neurons in a motor network may make distinct, but potentially complementary, contributions as either activators or specifiers of motor activity.

Expression2Kinases: mRNA profiling linked to multiple upstream regulatory layers.

MOTIVATION: Genome-wide mRNA profiling provides a snapshot of the global state of cells under different conditions. However, mRNA levels do not provide direct understanding of upstream regulatory mechanisms. Here, we present a new approach called Expression2Kinases (X2K) to identify upstream regulators likely responsible for observed patterns in genome-wide gene expression. By integrating chromatin immuno-precipitation (ChIP)-seq/chip and position weight matrices (PWMs) data, protein-protein interactions and kinase-substrate phosphorylation reactions, we can better identify regulatory mechanisms upstream of genome-wide differences in gene expression. We validated X2K by applying it to recover drug targets of food and drug administration (FDA)-approved drugs from drug perturbations followed by mRNA expression profiling; to map the regulatory landscape of 44 stem cells and their differentiating progeny; to profile upstream regulatory mechanisms of 327 breast cancer tumors; and to detect pathways from profiled hepatic stellate cells and hippocampal neurons. The X2K approach can advance our understanding of cell signaling and unravel drugs mechanisms of action. AVAILABILITY: The software and source code are freely available at: http://www.maayanlab.net/X2K. CONTACT: avi.maayan@mssm.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Coordination of distinct motor structures through remote axonal coupling of projection interneurons.

Complex behaviors often require coordinated movements of dissimilar motor structures. The underlying neural mechanisms are poorly understood. We investigated cycle-by-cycle coordination of two dissimilar feeding structures in Aplysia californica: the external lips and the internal radula. During feeding, the lips open while the radula protracts. Lip and radula motoneurons are located in the cerebral and buccal ganglia, respectively, and radula motoneurons are controlled by a well characterized buccal central pattern generator (CPG). Here, we examined whether the three electrically coupled lip motoneurons C15/16/17 are controlled by the buccal CPG or by a previously postulated cerebral CPG. Two buccal-cerebral projection interneurons, B34 and B63, which are part of the buccal CPG and mediate radula protraction, monosynaptically excite C15/16/17. Recordings from the B34 axon in the cerebral ganglion demonstrate its direct electrical coupling with C15/16/17, eliminating the need for a cerebral CPG. Moreover, when the multifunctional buccal CPG generates multiple forms of motor programs due to the activation of two inputs, the command-like neuron CBI-2 and the esophageal nerve (EN), C15/16 exhibit activity patterns that are distinct from C17. These distinct activity patterns result from combined activity of B34 and B63 and their differential excitation of C15/16 versus C17. In more general terms, we identified neuronal mechanisms that allow a single CPG to coordinate the phasing and activity of remotely located motoneurons innervating distinct structures that participate in the production of different motor outputs. We also demonstrated that axodendritic electrical coupling by projection interneurons plays a pivotal role in coordinating activity of these remotely located neurons.