Conserved roles for Hnf4 family transcription factors in zebrafish development and intestinal function.

Transcription factors play important roles in the development of the intestinal epithelium and its ability to respond to endocrine, nutritional, and microbial signals. Hepatocyte nuclear factor 4 family nuclear receptors are liganded transcription factors that are critical for the development and function of multiple digestive organs in vertebrates, including the intestinal epithelium. Zebrafish have 3 hepatocyte nuclear factor 4 homologs, of which, hnf4a was previously shown to mediate intestinal responses to microbiota in zebrafish larvae. To discern the functions of other hepatocyte nuclear factor 4 family members in zebrafish development and intestinal function, we created and characterized mutations in hnf4g and hnf4b. We addressed the possibility of genetic redundancy amongst these factors by creating double and triple mutants which showed different rates of survival, including apparent early lethality in hnf4a; hnf4b double mutants and triple mutants. RNA sequencing performed on digestive tracts from single and double mutant larvae revealed extensive changes in intestinal gene expression in hnf4a mutants that were amplified in hnf4a; hnf4g mutants, but limited in hnf4g mutants. Changes in hnf4a and hnf4a; hnf4g mutants were reminiscent of those seen in mice including decreased expression of genes involved in intestinal function and increased expression of cell proliferation genes, and were validated using transgenic reporters and EdU labeling in the intestinal epithelium. Gnotobiotics combined with RNA sequencing also showed hnf4g has subtler roles than hnf4a in host responses to microbiota. Overall, phenotypic changes in hnf4a single mutants were strongly enhanced in hnf4a; hnf4g double mutants, suggesting a conserved partial genetic redundancy between hnf4a and hnf4g in the vertebrate intestine

Reversibly switchable fluorescent Proteins for RESOLFT nanoscopy

Diffraction-limited lens-based optical microscopy fails to discern fluorescent features closer than ∼200 nm. All super-resolution microscopy (nanoscopy) approaches that fundamentally overcome the diffraction barrier rely on fluorophores that can adopt different states, typically a fluorescent ‘on-’state and a dark, non-fluorescent ‘off-’state. In reversible saturable optical linear fluorescence transitions (RESOLFT) nanoscopy, light is applied to induce transitions between two states and to switch fluorophores on and off at defined spatial coordinates. RESOLFT nanoscopy relies on metastable reversibly switchable fluorophores. Thereby, it is particularly suited for live-cell imaging, because it requires relatively low light levels to overcome the diffraction barrier. Most implementations of RESOLFT nanoscopy utilize reversibly photoswitchable fluorescent proteins (RSFPs), which are derivatives of proteins from the green fluorescent protein (GFP) family. In recent years, analysis of the molecular mechanisms of the switching processes have paved the way to a rational design of new RSFPs with superior characteristics for super-resolution microscopy. In this chapter, we focus on the newly developed RSFPs, the light-driven switching mechanisms and the use of RSFPs for RESOLFT nanoscopy