Phase separation and biorhythms control biological processes in the spatial and temporal dimensions, respectively, but mechanisms of four-dimensional integration remain elusive. Here, we identified an evolutionarily conserved XBP1s-SON axis that establishes a cell-autonomous mammalian 12-hour ultradian rhythm of nuclear speckle liquid-liquid phase separation (LLPS) dynamics, separate from both the 24-hour circadian clock and the cell cycle. Higher expression of nuclear speckle scaffolding protein SON, observed at early morning/early afternoon, generates diffuse and fluid nuclear speckles, increases their interactions with chromatin proactively, transcriptionally amplifies the unfolded protein response, and protects against proteome stress, whereas the opposites are observed following reduced SON level at early evening/late morning. Correlative Son and proteostasis gene expression dynamics are further observed across the entire mouse life span. Our results suggest that by modulating the temporal dynamics of proteostasis, the nuclear speckle LLPS may represent a previously unidentified (chrono)therapeutic target for pathologies associated with dysregulated proteostasis.

No abstract

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

13To understand how novel animal patterning emerged, one needs to ask how the 14 development of color patterns has changed among diverging species. Here we examine 15 three species of fruit flies -Drosophila guttifera (D. guttifera), Drosophila palustris (D. 16 palustris), and Drosophila subpalustris (D. subpalustris) -displaying a varying number 17 of abdominal spot rows that were either gained or lost throughout evolutionary time. 18 Through in situ hybridization, we examine the mRNA expression patterns for the 19 pigmentation genes Dopa decarboxylase (Ddc), tan (t), and yellow (y) during pupal 20 development. Our results show that Ddc, t, and y are co-expressed in identical patterns, 21 each foreshadowing the adult abdominal spots in D. guttifera, D. palustris, and D. 22 subpalustris. 23

No abstract