José Miguel Dias

DNA origami-based nanotechnology is a versatile tool for exploring fundamental biological questions and holds significant promise for future biomedical applications. Here, we leverage the optical transparency of the embryonic zebrafish to analyze live embryos injected intravenously with fluorescently labeled wireframe DNA origami nanosheets. Our approach integrated long-term, high-resolution imaging of transgenic live zebrafish embryos with single-cell RNA sequencing to elucidate the effects of oligolysine-polyethylene glycol copolymer (K-PEG) coating on the biodistribution of fluorescence signal in embryos injected with wireframe DNA origami nanosheets. We observed rapid accumulation of fluorescence signal in the caudal hematopoietic tissue (CHT). K-PEG coating mitigated the accumulation of fluorescence signal in CHT, enabling increased detection of signal in other tissues. Our findings highlighted the pivotal role of scavenger endothelial cells in DNA origami clearance, with K-PEG enabling the prolonged detection of fluorescence signal at the CHT. Furthermore, using a transgenic zebrafish line designed for targeted macrophage ablation, we found that macrophages contribute to the clearance of fluorescence signal in embryos injected with the noncoated but not with K-PEG-coated nanosheets. This study introduces a framework for the analyses of the biodistribution and clearance of DNA origami nanostructures in vivo with single-cell resolution in zebrafish models.

Skeletal muscle atrophy is a morbidity and mortality risk factor that happens with disuse, chronic disease, and aging. The tissue remodeling that happens during recovery from atrophy or injury involves changes in different cell types such as muscle fibers, and satellite and immune cells. Here, we show that the previously uncharacterized gene and protein Zfp697 is a damage-induced regulator of muscle remodeling. Zfp697/ZNF697 expression is transiently elevated during recovery from muscle atrophy or injury in mice and humans. Sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and extracellular matrix remodeling. Notably, although Zfp697 is expressed in several cell types in skeletal muscle, myofiber-specific Zfp697 genetic ablation in mice is sufficient to hinder the inflammatory and regenerative response to muscle injury, compromising functional recovery. We show that Zfp697 is an essential mediator of the interferon gamma response in muscle cells and that it functions primarily as an RNA-interacting protein, with a very high number of miRNA targets. This work identifies Zfp697 as an integrator of cell–cell communication necessary for tissue remodeling and regeneration.

Insulin binds the insulin receptor (IR) and regulates anabolic processes in target tissues. Impaired IR signalling is associated with multiple diseases, including diabetes, cancer and neurodegenerative disorders. IRs have been reported to form nanoclusters at the cell membrane in several cell types, even in the absence of insulin binding. Here we exploit the nanoscale spatial organization of the IR to achieve controlled multivalent receptor activation. To control insulin nanoscale spatial organization and valency, we developed rod-like insulin–DNA origami nanostructures carrying different numbers of insulin molecules with defined spacings. Increasing the insulin valency per nanostructure markedly extended the residence time of insulin–DNA origami nanostructures at the receptors. Both insulin valency and spacing affected the levels of IR activation in adipocytes. Moreover, the multivalent insulin design associated with the highest levels of IR activation also induced insulin-mediated transcriptional responses more effectively than the corresponding monovalent insulin nanostructures. In an in vivo zebrafish model of diabetes, treatment with multivalent—but not monovalent—insulin nanostructures elicited a reduction in glucose levels. Our results show that the control of insulin multivalency and spatial organization with nanoscale precision modulates the IR responses, independent of the insulin concentration. Therefore, we propose insulin nanoscale organization as a design parameter in developing new insulin therapies.

Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.

A Shh/Gli-driven incoherent feed-forward loop circuit encodes time and counterbalances noise in temporal neural patterning.

Skeletal muscle cells contain hundreds of myonuclei within a shared cytoplasm, presenting unique challenges for regulating gene expression. Certain transcriptional programs (e.g., postsynaptic machinery) are segregated to specialized domains, while others (e.g., contractile proteins) do not show spatial confinement. Furthermore, local stimuli, such as denervation, can induce transcriptional responses that are propagated along the muscle cells. Regulated transport of nuclear proteins (e.g., transcription factors) between myonuclei represents a potential mechanism for coordinating gene expression. However, the principles underlying the transport of nuclear proteins within multinucleated cells remain poorly defined. Here we used a mosaic transfection model to create myotubes that contained exactly one myonucleus expressing a fluorescent nuclear reporter and monitored its distribution among all myonuclei. We found that the transport properties of these model nuclear proteins in myotubes depended on molecular weight and nuclear import rate, as well as on myotube width. Interestingly, muscle hypertrophy increased the transport of high molecular weight nuclear proteins, while atrophy restricted the transport of smaller nuclear proteins. We have developed a mathematical model of nuclear protein transport within a myotube that recapitulates the results of our in vitro experiments. To test the relevance to nuclear proteins expressed in skeletal muscle, we studied the transport of two transcription factors—aryl hydrocarbon receptor nuclear translocator and sine oculis homeobox 1—and found that their distributions were similar to the reporter proteins with corresponding molecular weights. Together, these results define a set of variables that can be used to predict the spatial distributions of nuclear proteins within a myotube.

The new web resource EviNet provides an easily run interface to network enrichment analysis for exploration of novel, experimentally defined gene sets. The major advantages of this analysis are (i) applicability to any genes found in the global network rather than only to those with pathway/ontology term annotations, (ii) ability to connect genes via different molecular mechanisms rather than within one high-throughput platform, and (iii) statistical power sufficient to detect enrichment of very small sets, down to individual genes. The users' gene sets are either defined prior to upload or derived interactively from an uploaded file by differential expression criteria. The pathways and networks used in the analysis can be chosen from the collection menu. The calculation is typically done within seconds or minutes and the stable URL is provided immediately. The results are presented in both visual (network graphs) and tabular formats using jQuery libraries. Uploaded data and analysis results are kept in separated project directories not accessible by other users. EviNet is available at https://www.evinet.org/.

Cranial motor nerves in vertebrates are comprised of the three principal subtypes of branchial, visceral, and somatic motor neurons, which develop in typical patterns along the anteroposterior and dorsoventral axes of hindbrain. Here we demonstrate that the formation of branchial and visceral motor neurons critically depends on the transcription factors Nkx2.2 and Nkx2.9, which together determine the cell fate of neuronal progenitor cells. Disruption of both genes in mouse embryos results in complete loss of the vagal and spinal accessory motor nerves, and partial loss of the facial and glossopharyngeal motor nerves, while the purely somatic hypoglossal and abducens motor nerves are not diminished. Cell lineage analysis in a genetically marked mouse line reveals that alterations of cranial nerves in Nkx2.2; Nkx2.9 double-deficient mouse embryos result from changes of cell fate in neuronal progenitor cells. As a consequence progenitors of branchiovisceral motor neurons in the ventral p3 domain of hindbrain are transformed to somatic motor neurons, which use ventral exit points to send axon trajectories to their targets. Cell fate transformation is limited to the caudal hindbrain, as the trigeminal nerve is not affected in double-mutant embryos suggesting that Nkx2.2 and Nkx2.9 proteins play no role in the development of branchiovisceral motor neurons in hindbrain rostral to rhombomere 4.