Csu x

eGFP fusion constructs were imaged at Z-series stacks of 0.2 µm, acquired at 37°C and 5% CO₂, on a Yokogawa CSU-X spinning disk confocal, using a 60× 1.2 NA water immersion objective. An Andor iXon+ EMCCD camera was used to acquire images for the emission of the eGFP (488-nm laser line). The colocalization of transcription factors with different DNA regions was analyzed with an image segmentation pipeline in Fiji. Cell nuclei were identified and segmented by K-means clustering based on the Hoechst signal. Three regions with high, medium, and low Hoechst levels within each nucleus were defined, respectively representing heterochromatic, DNA-dense, and DNA-poor regions. Subsequently, the corresponding eGFP signal intensity in each of the three segmented regions was measured. Data are represented in log₂ values in which the intensity of GFP in each DNA region was divided by the whole-cell intensity. At least 20 cells were analyzed per condition. Due to the quality of the Hoechst staining, DNA segmentation by K means clustering resulted in some variability among cells. Thus, only cells yielding a predefined segmentation ratio among the three different regions—heterochromatin (10%–20%), DNA-dense (40%–60%), and DNA-poor (40%)—were included in the final analysis. A max projection of the 20–30 0.2-µm Z slices that were acquired was applied before DNA segmentation.

Cells were imaged on an inverted microscope (Ti-E; Nikon, Melville, NY) with a confocal scanhead (CSU-X; Yokogawa Electric, Musashino, Tokyo, Japan); laser merge module containing 491, 561, and 642 laser lines (Spectral Applied Research, Richmond Hill, Ontario, Canada); and an Andor Zyla scientific complementary metal-oxide-semiconductor camera (Belfast, Northern Ireland, UK). METAMORPH acquisition software (Molecular Devices, Eugene, OR) was used to control the microscope hardware. Images were acquired using a 40× 1 NA Plan Apo oil-immersion objective. Samples were mounted on a live imaging chamber (Chamlide, Seoul, Korea) and maintained at 37°C. For live cell imaging, DMEM was supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

2 dpf morpholino-injected WT AB and mpx:mCherry or 3 dpf WT and mydgf^−/− mpx:mCherry zebrafish larvae were wounded by tail transection or thermal injury, or injected with zebrafish MYDGF protein in the otic vesicle, as described above. Larvae were fixed in 4% PFA at 4°C at 1 and 6 hpw (following tail transection), 3 and 24 hpb (following burn wound) or 2 h after otic vesicle injection. Caudal fins were imaged in PBS at room temperature on a Zeiss Zoomscope (EMS3/SyCoP3; 1× Plan-NeoFluar Z objective; Zeiss) with an Axiocam Mrm charge-coupled device camera using ZenPro 2012 software (Zeiss). For protein injection assays, images of the otic vesicle region were acquired in PBS at room temperature on a spinning-disk confocal (CSU-X; Yokogawa) on a Zeiss Observer Z.1 inverted microscope and an electron-multiplying charge-coupled device Evolve 512 camera (Photometrics), with a Plan-Apochromat 20×/NA 0.8 air objective (5-µm optical sections, 2,355 × 512 resolution) using ZenPro 2012 software (Zeiss). Neutrophil numbers were counted manually in z-projected images using Zen 2.3 Lite software (Zeiss). Neutrophil numbers were counted in the area distal to the tip of the notochord (tail transections), the area distal to the caudal vessel loop (burn wounds), or within the confines of the otic vesicle (protein injections).