DOI: 10.17863/cam.47583
title: Additional Materials of the Thesis "Decoding the Notch signal: investigating dynamics of transcription directed by Notch responsive enhancers". Julia Falo Sanjuan, January 2020

description: Movies and plasmid sequences described in the Thesis "Activity and specificity of Notch regulated enhancers". Julia Falo Sanjuan, January 2020 Time-lapse confocal microscopy of Drosophila embryos in which transcription from Notch responsive was imaged live. Movie 01 - Expression of m5/m8. Movie showing transcription from m5/m8-peve in the mesectoderm stripe during nc14. Also note earlier, Notch independent transcription in broad domains in nc10-13 and in few scattered cells during the first few minutes of nc14, followed by a long period (approximately 20 min) of inactivity before MSE stripe cells initiate transcription. Maximum intensity projection (19x1\textmu m stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 10s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 02 - Expression of sim. Movie showing transcription from sim-peve in the mesectoderm stripe and some mesodermal cells during nc14. Also note activity in scattered cells before nc14. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 03 - Ectopic expression of m5/m8 with eve2-NICD. Movie showing ectopic transcription from m5/m8-peve in the eve2 domain during nc14. Maximum intensity projection (29x1\textmu m stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 04 - Ectopic expression of sim with eve2-NICD. Movie showing ectopic transcription from sim-peve in the eve2 domain during nc14. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 05 - Regions of ectopic expression of m5/m8 and sim with eve2-NICD. Combined movie of m5/m8-peve (left) and sim-peve (right) showing ectopic transcription in the eve2 stripe. The maximum projection of the MCP-GFP signal is overlaid with tracked nuclei false colored with the maximum intensity pixel in each nucleus. Active nuclei in each of the analyzed regions is marked with a different color: red (mesoderm), purple (mesectoderm), blue (neuroectoderm) and green (dorsal ectoderm). Anterior to the left; embryo imaged from the ventral side. Movie 06 - Transcription of two m5/m8-peve MS2 reporters in the presence of ectopic NICD. Movie showing ectopic transcription from m5/m8-peve x2 in the eve2 domain during nc14. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 07 - Changes in nuclear size and shape during nc14. Movie showing maximum projection of medial slices and orthogonal views of Nup107-GFP. 0.18 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 08 - Expression of m5/m8 starts during cellularization. Movie showing cellularizing membranes using the marker Gap43-mCherry (maximum intensity projection of medial slices and orthogonal views, left) and transcription from m5/m8-peve (maximum intensity projection (29x1um stacks) of the MCP-GFP channel, right). 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 09 - Localization of Notch and Delta during cellularization. Movies showing maximum projection of medial slices and orthogonal views of Ni-GFP (left) and Dl-mScarlet (right). 0.36 um/px XY resolution and final time resolution of 60s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 10 - Expression of vnd-peve. Movie showing transcription from vnd-peve in the neuroectoderm. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 11 - Transcription of m5/m8 MS2 in a fog mutant background. Mesoderm invagination is delayed and does not proceed properly, resulting in a folded gastrulation phenotype. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Plasmid sequences - annotated sequences of all generated plasmids: pattB-m5/m8-peve-MS2-LacZ pattB-m5/m8-hsp70-MS2-LacZ pattB-m5/m8-pm5-MS2-LacZ pattB-m5/m8-pm6-MS2-LacZ pattB-m5/m8-pm7-MS2-LacZ pattB-m5/m8-pm8-MS2-LacZ pattB-m5/m8-psimE-MS2-LacZ pattB-sim-peve-MS2-LacZ pattB-sim-psimE-MS2-LacZ pattB-m8NE-peve-MS2-LacZ pattB-m8NE-pm8-MS2-LacZ pattB-vnd-peve-MS2-LacZ pattB-pUbi-MS2-LacZ pattB-m5/m8_insSPS-peve-MS2-LacZ pattB-sim_SPS-peve-MS2-LacZ pattB-m5/m8_Dtwi-peve-MS2-LacZ pattB-m5/m8_Ddl-peve-MS2-LacZ pattB-m5/m8_DtwiDdl-peve-MS2-LacZ pCR4-E-m5m8 pCR4-NE-sim pCR4-NE-m8NE' pCR4-E-m5m8_insSPS pCR4-NE-sim_SPS pCR4-E-m5m8_Ddl pCR4-E-m5m8_Dtwi pCR4-E-m5m8_DtwiDdl pattB-eve2x2-peve-FRT-STOP-FRT-NICD-eve3'UTR