Dynamics of miRNA accumulation during C. elegans larval development.

Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

H3K9me selectively blocks transcription factor activity and ensures differentiated tissue integrity.

The developmental role of histone H3K9 methylation (H3K9me), which typifies heterochromatin, remains unclear. In Caenorhabditis elegans, loss of H3K9me leads to a highly divergent upregulation of genes with tissue and developmental-stage specificity. During development H3K9me is lost from differentiated cell type-specific genes and gained at genes expressed in earlier developmental stages or other tissues. The continuous deposition of H3K9me2 by the SETDB1 homolog MET-2 after terminal differentiation is necessary to maintain repression. In differentiated tissues, H3K9me ensures silencing by restricting the activity of a defined set of transcription factors at promoters and enhancers. Increased chromatin accessibility following the loss of H3K9me is neither sufficient nor necessary to drive transcription. Increased ATAC-seq signal and gene expression correlate at a subset of loci positioned away from the nuclear envelope, while derepressed genes at the nuclear periphery remain poorly accessible despite being transcribed. In conclusion, H3K9me deposition can confer tissue-specific gene expression and maintain the integrity of terminally differentiated muscle by restricting transcription factor activity.

Resolving Neurodevelopmental and Vision Disorders Using Organoid Single-Cell Multi-omics.

Human organoid models of the central nervous system, including the neural retina, are providing unprecedented opportunities to explore human neurodevelopment and neurodegeneration in controlled culture environments. In this Perspective, we discuss how the single-cell multi-omic toolkit has been used to identify features and limitations of brain and retina organoids and how these tools can be deployed to study congenital brain malformations and vision disorders in organoids. We also address how to improve brain and retina organoid protocols to revolutionize in vitro disease modeling.

An interplay of miRNA abundance and target site architecture determines miRNA activity and specificity.

MicroRNAs often occur in families whose members share an identical 5' terminal 'seed' sequence. The seed is a major determinant of miRNA activity, and family members are thought to act redundantly on target mRNAs with perfect seed matches, i.e. sequences complementary to the seed. However, recently sequences outside the seed were reported to promote silencing by individual miRNA family members. Here, we examine this concept and the importance of miRNA specificity for the robustness of developmental gene control. Using the let-7 miRNA family in Caenorhabditis elegans, we find that seed match imperfections can increase specificity by requiring extensive pairing outside the miRNA seed region for efficient silencing and that such specificity is needed for faithful worm development. In addition, for some target site architectures, elevated miRNA levels can compensate for a lack of complementarity outside the seed. Thus, some target sites require higher miRNA concentration for silencing than others, contrasting with a traditional binary distinction between functional and non-functional sites. We conclude that changing miRNA concentrations can alter cellular miRNA target repertoires. This diversifies possible biological outcomes of miRNA-mediated gene regulation and stresses the importance of target validation under physiological conditions to understand miRNA functions in vivo.

Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis.