Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling.

Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors (TFs), such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C. robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2.b and an inferred ATTA-binding transcriptional cofactor. Here, we show that the homeobox TF Lhx3/4 serves as the lineage-determining TF that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings, we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term "cofactor-dependent induction." Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.

Examining the evidence for extracellular RNA function in mammals.

The presence of RNAs in the extracellular milieu has sparked the hypothesis that RNA may play a role in mammalian cell-cell communication. As functional nucleic acids transfer from cell to cell in plants and nematodes, the idea that mammalian cells also transfer functional extracellular RNA (exRNA) is enticing. However, untangling the role of mammalian exRNAs poses considerable experimental challenges. This Review discusses the evidence for and against functional exRNAs in mammals and their proposed roles in health and disease, such as cancer and cardiovascular disease. We conclude with a discussion of the forward-looking prospects for studying the potential of mammalian exRNAs as mediators of cell-cell communication.

Elimination of Calm1 long 3'-UTR mRNA isoform by CRISPR-Cas9 gene editing impairs dorsal root ganglion development and hippocampal neuron activation in mice.

The majority of mouse and human genes are subject to alternative cleavage and polyadenylation (APA), which most often leads to the expression of two or more alternative length 3' untranslated region (3'-UTR) mRNA isoforms. In neural tissues, there is enhanced expression of APA isoforms with longer 3'-UTRs on a global scale, but the physiological relevance of these alternative 3'-UTR isoforms is poorly understood. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that generates short (Calm1-S) and long (Calm1-L) 3'-UTR mRNA isoforms via APA. We found Calm1-L expression to be largely restricted to neural tissues in mice including the dorsal root ganglion (DRG) and hippocampus, whereas Calm1-S was more broadly expressed. smFISH revealed that both Calm1-S and Calm1-L were subcellularly localized to neural processes of primary hippocampal neurons. In contrast, cultured DRG showed restriction of Calm1-L to soma. To investigate the in vivo functions of Calm1-L, we implemented a CRISPR-Cas9 gene editing strategy to delete a small region encompassing the Calm1 distal poly(A) site. This eliminated Calm1-L expression while maintaining expression of Calm1-S Mice lacking Calm1-L (Calm1ΔL/ΔL ) exhibited disorganized DRG migration in embryos, and reduced experience-induced neuronal activation in the adult hippocampus. These data indicate that Calm1-L plays functional roles in the central and peripheral nervous systems.

Global accumulation of circRNAs during aging in Caenorhabditis elegans.

BACKGROUND: Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5' and 3' ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues. RESULTS: Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated a high-confidence set of 1166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by RT-qPCR. The expression of 459 circRNAs was determined to be distinct from the expression of linear RNAs from the same host genes, demonstrating host gene independence of circRNA age-accumulation. CONCLUSIONS: We attribute the global scale of circRNA age-accumulation to the high composition of post-mitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine whether there are functional consequences of circRNA accumulation during aging.

SARS-CoV-2 ORF3A interacts with the Clic-like chloride channel-1 (CLCC1) and triggers an unfolded protein response.

Understanding the interactions between SARS-CoV-2 and host cell machinery may reveal new targets to treat COVID-19. We focused on an interaction between the SARS-CoV-2 ORF3A accessory protein and the CLIC-like chloride channel-1 (CLCC1). We found that ORF3A partially co-localized with CLCC1 and that ORF3A and CLCC1 could be co-immunoprecipitated. Since CLCC1 plays a role in the unfolded protein response (UPR), we hypothesized that ORF3A may also play a role in the UPR. Indeed, ORF3A expression triggered a transcriptional UPR that was similar to knockdown of CLCC1. ORF3A expression in 293T cells induced cell death and this was rescued by the chemical chaperone taurodeoxycholic acid (TUDCA). Cells with CLCC1 knockdown were partially protected from ORF3A-mediated cell death. CLCC1 knockdown upregulated several of the homeostatic UPR targets induced by ORF3A expression, including HSPA6 and spliced XBP1, and these were not further upregulated by ORF3A. Our data suggest a model where CLCC1 silencing triggers a homeostatic UPR that prevents cell death due to ORF3A expression.

Robo1 and 2 Repellent Receptors Cooperate to Guide Facial Neuron Cell Migration and Axon Projections in the Embryonic Mouse Hindbrain.

The facial nerve is necessary for our ability to eat, speak, and make facial expressions. Both the axons and cell bodies of the facial nerve undergo a complex embryonic developmental pattern involving migration of the cell bodies caudally and tangentially through rhombomeres, and simultaneously the axons projecting to exit the hindbrain to form the facial nerve. Our goal in this study was to test the functions of the chemorepulsive receptors Robo1 and Robo2 in facial neuron migration and axon projection by analyzing genetically marked motor neurons in double-mutant mouse embryos through the migration time course, E10.0-E13.5. In Robo1/2 double mutants, axon projection and cell body migration errors were more severe than in single mutants. Most axons did not make it to their motor exit point, and instead projected into and longitudinally within the floor plate. Surprisingly, some facial neurons had multiple axons exiting and projecting into the floor plate. At the same time, a subset of mutant facial cell bodies failed to migrate caudally, and instead either streamed dorsally toward the exit point or shifted into the floor plate. We conclude that Robo1 and Robo2 have redundant functions to guide multiple aspects of the complex cell migration of the facial nucleus, as well as regulating axon trajectories and suppressing formation of ectopic axons.

ISL1-based LIM complexes control Slit2 transcription in developing cranial motor neurons.

LIM-homeodomain (HD) transcription factors form a multimeric complex and assign neuronal subtype identities, as demonstrated by the hexameric ISL1-LHX3 complex which gives rise to somatic motor (SM) neurons. However, the roles of combinatorial LIM code in motor neuron diversification and their subsequent differentiation is much less well understood. In the present study, we demonstrate that the ISL1 controls postmitotic cranial branchiomotor (BM) neurons including the positioning of the cell bodies and peripheral axon pathfinding. Unlike SM neurons, which transform into interneurons, BM neurons are normal in number and in marker expression in Isl1 mutant mice. Nevertheless, the movement of trigeminal and facial BM somata is stalled, and their peripheral axons are fewer or misrouted, with ectopic branches. Among genes whose expression level changes in previous ChIP-seq and microarray analyses in Isl1-deficient cell lines, we found that Slit2 transcript was almost absent from BM neurons of Isl1 mutants. Both ISL1-LHX3 and ISL1-LHX4 bound to the Slit2 enhancer and drove endogenous Slit2 expression in SM and BM neurons. Our findings suggest that combinations of ISL1 and LHX factors establish cell-type specificity and functional diversity in terms of motor neuron identities and/or axon development.