Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process.

Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the ß-actin and lens differentiation-specific α-, ß-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.

A multicolor podocyte reporter highlights heterogeneous podocyte changes in focal segmental glomerulosclerosis.

In contrast to most glomerular diseases, the injury pattern in focal segmental glomerulosclerosis (FSGS) is highly heterogeneous, even though podocytes are genetically identical and exposed to the same environmental factors. To understand changes in individual podocytes, we generated and analyzed a stochastic multicolor Cre-reporter, encoding four fluorescent proteins. In these animals, podocytes were randomly labeled allowing individual cells and their foot processes to be distinguished. In healthy animals, podocyte size and structure showed little cell to cell variability. In the doxorubicin-induced FSGS model, fluorescent-labeled glomerular podocyte numbers decreased and fluorescent cells could be recovered from the urine. The size of the remaining podocytes showed a high degree of heterogeneity, some cells remained small, while others enlarged. Both enlarged and non-enlarged podocytes showed alterations in their foot process morphology. Thus, by the virtue of a multicolor cre-reporter, individual podocytes could be viewed in real time at a cellular resolution indicating a heterogeneous podocyte injury response during the pathogenesis of FSGS.