Tissue-specific targeting of DNA nanodevices in a multicellular living organism

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology

Modular Organization of Cis-regulatory Control Information of Neurotransmitter Pathway Genes in Caenorhabditis elegans

Here, Serrano-Saiz et al. describe the cis-regulatory logic of how neurotransmitter identity is imposed onto individual, distinct neuron types... We explore here the cis-regulatory logic that dictates gene expression in specific cell types in the nervous system. We focus on a set of eight genes involved in the synthesis, transport, and breakdown of three neurotransmitter systems: acetylcholine (unc-17/VAChT, cha-1/ChAT, cho-1/ChT, and ace-2/AChE), glutamate (eat-4/VGluT), and γ-aminobutyric acid (unc-25/GAD, unc-46/LAMP, and unc-47/VGAT). These genes are specifically expressed in defined subsets of cells in the nervous system. Through transgenic reporter gene assays, we find that the cellular specificity of expression of all of these genes is controlled in a modular manner through distinct cis-regulatory elements, corroborating the previously inferred piecemeal nature of specification of neurotransmitter identity. This modularity provides the mechanistic basis for the phenomenon of “phenotypic convergence,” in which distinct regulatory pathways can generate similar phenotypic outcomes (i.e., the acquisition of a specific neurotransmitter identity) in different neuron classes. We also identify cases of enhancer pleiotropy, in which the same cis-regulatory element is utilized to control gene expression in distinct neuron types. We engineered a cis-regulatory allele of the vesicular acetylcholine transporter, unc-17/VAChT, to assess the functional contribution of a “shadowed” enhancer. We observed a selective loss of unc-17/VAChT expression in one cholinergic pharyngeal pacemaker motor neuron class and a behavioral phenotype that matches microsurgical removal of this neuron. Our analysis illustrates the value of understanding cis-regulatory information to manipulate gene expression and control animal behavior.This work was supported by the Howard Hughes Medical Institute. E.S.-S. has been supported by the Ramon y Cajal program (RYC-2016-20537), and M.G. was supported by the European Molecular Biology Organization and Human Frontier Science Program postdoctoral fellowships.Peer reviewe

Τα μοριακά μονοπάτια Notch and NF-kB στην καρδιακή ανάπτυξη και ασθένεια

Congenital heart disease is the commonest human birth defect occurring in 1% of the population worldwide, whereas acquired heart disease is the main cause of mortality in the elderly western populations, highlighting the importance of research focus on signaling pathways involved in cardiac development and disease. Notch signaling pathway is important for cell-cell communication that controls tissue formation and homeostasis during embryonic and adult life, but the precise cell targets of Notch signaling in the mammalian heart remain poorly defined. Here, we report that conditional over-expression of Notch1 intracellular domain (NICD1) in the embryonic cardiomyocyte compartment results in developmental defects and perinatal lethality in mice. In contrast, augmentation of endogenous Notch reactivation after myocardial infarction in the adult by intramyocardial delivery of a Notch1 pseudoligand increases survival rate improves cardiac functional performance and minimizes fibrosis, promoting proliferative and angiogenic mechanisms. These results reveal a strict requirement for cell-autonomous modulation of Notch signaling during heart morphogenesis, and illustrate how the same signaling pathway that promotes congenital heart defects when perturbed in the embryo can be therapeutically redeployed for the treatment of adult myocardial damage. Additionally, we sought to determine the role of NF-κB pathway in the adult myocardium and acquired cardiac disease. Insight into the function of nuclear factor κB (NF-κB) in the adult heart has been hampered by the embryonic lethality of constitutive NF-κB inactivation in mice. Using Cre/loxP technology, we disrupted in a cardiac-specific manner the NF-κB essential modulator (NEMO) in the murine heart, which simulated the adaptive changes underlying human heart failure, causing adultonset dilated cardiomyopathy accompanied by inflammation and apoptosis. Pressure overload challenges of NF-κB-deficient young hearts precociously induced the functional decrements that develop spontaneously in older knockout animals. Oxidative stress in NF-κB-deficient cardiomyocytes is a critical pathological component that can be attenuated with antioxidant diet in vivo. These results reveal an essential physiological role for NF-κB in the adult heart to maintain cardiac function in response to aging-related or mechanical challenges, in part through modulation of oxidative stress. Taken together, our observations provide valuable insights regarding the cardiomyocyteautonomous role of Notch1 and NEMO/ NF-κB signaling in congenital and acquired heart disease.Η συγγενής καρδιοπάθεια αποτελεί την πιο συχνή μορφή ανθρώπινων δυσπλασιών (1% του παγκόσμιου πληθυσμού), ενώ η επίκτητη καρδιοπάθεια είναι η κύρια αιτία θνησιμότητας στον δυτικό ηλικιωμένο πληθυσμό, υπογραμμίζοντας την σημαντικότητα ερευνητικών προσπαθειών εστιαζόμενων στην μελέτη μοριακών μονοπατιών που εμπλέκονται στην καρδιακή ανάπτυξη και ασθένεια. Το μοριακό μονοπάτι Notch είναι απαραίτητο για μορφές διακυτταρικής επικοινωνίας, οι οποίες επηρεάζουν τον σχηματισμό και ομοιοστασία ιστών κατά την διάρκεια της εμβρυικής και ενήλικης ζωής. Ωστόσο, δεν είναι σαφές σε ποιους κυτταρικούς πληθυσμούς της καρδιάς θηλαστικών το μοριακό μονοπάτι Notch είναι ενεργό. Εδώ αναφέρουμε ότι υπερ-έκφραση του ενδοκυτταρικού τμήματος του Notch1 υποδοχέα (NICD1) σε μυοκύτταρα της εμβρυικής καρδιάς οδηγεί σε αναπτυξιακές ανωμαλίες και πρόωρη θνησιμότητα. Αντιθέτως, ενίσχυση της ενδογενούς Notch ενεργοποίησης μετά από έμφραγμα του μυοκαρδίου με την χρήση ενός ψευδοπροσδέματος (pseudoligand) για τον υποδοχέα Notch1 μειώνει το ποσοστό θνησιμότητας, βελτιώνει την καρδιακή λειτουργία και ελαχιστοποιεί την εναπόθεση κολλαγόνου (ίνωση), προάγωντας μηχανισμούς κυτταρικού πολλαπλασιασμού και δημιουργία νέων αιμοφόρων αγγείων. Τα αποτελέσματα μας αποκαλύπτουν πως ο βαθμός ενεργότητας του Notch μοριακού μονοπατιού στα καρδιομυοκύτταρα επηρεάζει την καρδιακή ανάπτυξη και αποδεικνύουν πως το ίδιο μοριακό μονοπάτι το οποίο προκαλεί καρδιακές ανωμαλίες όταν επηρεαστεί κατά την εμβρυική ανάπτυξη, μπορεί να χρησιμοποιηθεί για θεραπεία ασθενών με έμφραγμα του μυοκαρδίου. Επίσης, μελετήσαμε τον ρόλο του μεταγραφικού παράγοντα nuclear factor κB (NF- κB) στο μυοκάρδιο στα πλαίσια της επίκτητης καρδιακής ασθένειας. Οι μοριακές δράσεις του NF- κB στην ενήλικη καρδιά παραμένουν άγνωστες διότι η καθολική απενεργοποίηση του NF-κB σε ποντίκια οδηγεί σε πρόωρη θνησιμότητα κατά την εμβρυική ανάπτυξη. Χρησιμοποιώντας τεχνολογία Cre/loxP, απενεργοποιήσαμε το γονίδιο Νemo (NF-κB essential modulator) ειδικά στα καρδιομυοκύτταρα ποντικών. Η συγκεκριμένη προσέγγιση οδήγησε σε ανάπτυξη καρδιακής φλεγμονής και αποπτώσης, οι οποίες οδήγησαν σε καρδιομυοπάθεια κατά το ενήλικο στάδιο ζωής. Επιπρόσθετα, πειράματα επαγωγής υπερτροφίας σε νεαρές καρδιές ποντικών με μεταλλαγμένο το Νemo γονίδιο οδήγησαν πρόωρα σε καρδιακή δυσλειτουργία, η οποία θυμίζει την σποραδική καρδιοπάθεια που παρατηρείται σε ενήλικα μεταλλαγμένα Νemo ποντίκια. Πιστεύουμε πως η απώλεια του Νemo γονιδίου στα καρδιομυοκύτταρα οδηγεί σε οξειδωτική δυσλειτουργία η οποία προκαλεί καρδιομυοπάθεια, διότι αντιοξειδωτική δίαιτα επέφερε βελτίωση της καρδιακής παθολογίας στα μεταλλαγμένα Νemo ποντίκια. Τα αποτελέσματα μας αποκαλύπτουν έναν απαραίτητο ρόλο για τον μεταγραφικό παράγοντα NF-κB στην ενήλικη καρδιά ώστε να διατηρεί την φυσιολογική της λειτουργία ανταποκρινόμενη σε μηχανικές επιβαρύνσεις ή γήρανση. Συνοψίζοντας, οι παρατηρήσεις μας συνεισφέρουν στην βαθύτερη κατανόηση του ρόλου των μοριακών μονοπατιών Notch1 και NF-κB στα καρδιομυοκύτταρα στα πλαίσια συγγενών και επίκτητων καρδιακών ανωμαλιών

Protocol for auxin-inducible protein degradation in <i>C. elegans</i> using different auxins and TIR1-expressing strains

The auxin-inducible degron (AID) system is a powerful tool to deplete proteins in vivo. Here, we present a protocol for AID-mediated depletion of two proteins (CFI-1/AT-rich interaction domain 3 [ARID3] and Y47D3A.21/density-regulated re-initiation and release factor [DENR]) in C. elegans tissues using different auxins and transport inhibitor response 1 (TIR1)-expressing strains. We describe steps for genetic crossing, sample preparation, fluorescent microscopy, and treatment with either natural (indole-3-acetic acid [IAA]) or synthetic (1-naphthaleneacetic acid, potassium salt [K-NAA]) auxins. We then detail procedures for comparing the degree of CFI-1 depletion in C. elegans neurons upon panneuronal or pansomatic TIR1 expression. For complete details on the use and execution of this protocol, please refer to Li et al.</p

Coordinated regulation of cholinergic motor neuron traits through a conserved terminal selector gene

Cholinergic motor neurons are defined by the coexpression of a battery of genes encoding proteins that act sequentially to synthesize, package and degrade acetylcholine and reuptake its breakdown product, choline. How expression of these critical motor neuron identity determinants is controlled and coordinated is not understood. We show here that, in the nematode Caenorhabditis elegans, all members of the cholinergic gene battery, as well as many other markers of terminal motor neuron fate, are co-regulated by a shared cis-regulatory signature and a common trans-acting factor, the phylogenetically conserved COE (Collier, Olf, EBF)-type transcription factor UNC-3. UNC-3 initiated and maintained expression of cholinergic fate markers and was sufficient to induce cholinergic fate in other neuron types. UNC-3 furthermore operated in negative feedforward loops to induce the expression of transcription factors that repress individual UNC-3-induced terminal fate markers, resulting in diversification of motor neuron differentiation programs in specific motor neuron subtypes. A chordate ortholog of UNC-3, Ciona intestinalis COE, was also both required and sufficient for inducing a cholinergic fate. Thus, UNC-3 is a terminal selector for cholinergic motor neuron differentiation whose function is conserved across phylogeny

Control of spinal motor neuron terminal differentiation through sustained <i>Hoxc8</i> gene activity

Spinal motor neurons (MNs) constitute cellular substrates for several movement disorders. Although their early development has received much attention, how spinal MNs become and remain terminally differentiated is poorly understood. Here, we determined the transcriptome of mouse MNs located at the brachial domain of the spinal cord at embryonic and postnatal stages. We identified novel transcription factors (TFs) and terminal differentiation genes (e.g. ion channels, neurotransmitter receptors, adhesion molecules) with continuous expression in MNs. Interestingly, genes encoding homeodomain TFs (e.g. HOX, LIM), previously implicated in early MN development, continue to be expressed postnatally, suggesting later functions. To test this idea, we inactivated Hoxc8 at successive stages of mouse MN development and observed motor deficits. Our in vivo findings suggest that Hoxc8 is not only required to establish, but also maintain expression of several MN terminal differentiation markers. Data from in vitro generated MNs indicate Hoxc8 acts directly and is sufficient to induce expression of terminal differentiation genes. Our findings dovetail recent observations in Caenorhabditis elegans MNs, pointing toward an evolutionarily conserved role for Hox in neuronal terminal differentiation

Transcriptional Coordination of Synaptogenesis and Neurotransmitter Signaling

SummaryDuring nervous system development, postmitotic neurons face the challenge of generating and structurally organizing specific synapses with appropriate synaptic partners. An important unexplored question is whether the process of synaptogenesis is coordinated with the adoption of specific signaling properties of a neuron. Such signaling properties are defined by the neurotransmitter system that a neuron uses to communicate with postsynaptic partners, the neurotransmitter receptor type used to receive input from presynaptic neurons, and, potentially, other sensory receptors that activate a neuron. Elucidating the mechanisms that coordinate synaptogenesis, neuronal activation, and neurotransmitter signaling in a postmitotic neuron represents one key approach to understanding how neurons develop as functional units. Using the SAB class of Caenorhabditis elegans motor neurons as a model system, we show here that the phylogenetically conserved COE-type transcription factor UNC-3 is required for synaptogenesis. UNC-3 directly controls the expression of the ADAMTS-like protein MADD-4/Punctin, a presynaptically secreted synapse-organizing molecule that clusters postsynaptic receptors. UNC-3 also controls the assembly of presynaptic specializations and ensures the coordinated expression of enzymes and transporters that define the cholinergic neurotransmitter identity of the SAB neurons. Furthermore, synaptic output properties of the SAB neurons are coordinated with neuronal activation and synaptic input, as evidenced by UNC-3 also regulating the expression of ionotropic neurotransmitter receptors and putative stretch receptors. Our study shows how synaptogenesis and distinct, function-defining signaling features of a postmitotic neuron are hardwired together through coordinated transcriptional control

Multiple congenital malformations of Wolf-Hirschhorn syndrome are recapitulated in Fgfrl1 null mice

Wolf-Hirschhorn syndrome (WHS) is caused by deletions in the short arm of chromosome 4 (4p) and occurs in about one per 20,000 births. Patients with WHS display a set of highly variable characteristics including craniofacial dysgenesis, mental retardation, speech problems, congenital heart defects, short stature and a variety of skeletal anomalies. Analysis of patients with 4p deletions has identified two WHS critical regions (WHSCRs); however, deletions targeting mouse WHSCRs do not recapitulate the classical WHS defects, and the genes contributing to WHS have not been conclusively established. Recently, the human FGFRL1 gene, encoding a putative fibroblast growth factor (FGF) decoy receptor, has been implicated in the craniofacial phenotype of a WHS patient. Here, we report that targeted deletion of the mouse Fgfrl1 gene recapitulates a broad array of WHS phenotypes, including abnormal craniofacial development, axial and appendicular skeletal anomalies, and congenital heart defects. Fgfrl1 null mutants also display a transient foetal anaemia and a fully penetrant diaphragm defect, causing prenatal and perinatal lethality. Together, these data support a wider role for Fgfrl1 in development, implicate FGFRL1 insufficiency in WHS, and provide a novel animal model to dissect the complex aetiology of this human disease

A terminal selector prevents a Hox transcriptional switch to safeguard motor neuron identity throughout life

To become and remain functional, individual neuron types must select during development and maintain throughout life their distinct terminal identity features, such as expression of specific neurotransmitter receptors, ion channels and neuropeptides. Here, we report a molecular mechanism that enables cholinergic motor neurons (MNs) in the C. elegans ventral nerve cord to select and maintain their unique terminal identity. This mechanism relies on the dual function of the conserved terminal selector UNC-3 (Collier/Ebf). UNC-3 synergizes with LIN-39 (Scr/ Dfd/Hox4-5) to directly co-activate multiple terminal identity traits specific to cholinergic MNs, but also antagonizes LIN-39’s ability to activate terminal features of alternative neuronal identities. Loss of unc-3 causes a switch in the transcriptional targets of LIN-39, thereby alternative, not cholinergic MN-specific, terminal features become activated and locomotion defects occur. The strategy of a terminal selector preventing a transcriptional switch may constitute a general principle for safeguarding neuronal identity throughout life