| Experiment Id | GSE95108 | Name | miR-205 is a critical regulator of lacrimal gland development |
| Experiment Type | RNA-Seq | Study Type | WT vs. Mutant |
| Source | GEO | Curation Date | 2023-12-06 |
| description | Tears are essential for the maintenance of the terrestrial animal ocular surface and the lacrimal gland is the source of the aqueous layer of the tear film. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an essential gene for lacrimal gland development. Knockout mice lacking miR-205 fail to develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal bud initiation. RNA-seq analysis uncovered several up-regulated miR-205 targets, including Inppl1, a negative regulator of Akt signaling. Data indicate that Akt signaling is required within lacrimal gland epithelia and is activated by Fgf10. Furthermore, combinatorial epistatic deletion of Fgf10 and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways downstream of Fgf10 to regulate glandular development. These data show that a single microRNA is a key regulator for lacrimal gland initiation in mice and highlights the important role of microRNAs during organogenesis. Laser capture and RNA isolation. The laser capture staining protocol was adapted from the Laser Capture Molecular Core at Ohio State University. Briefly, E12.5 embryo heads were dissected and flash frozen in OCT for cyrosectioning. Sections were collected at 10 µM onto RNase-Zap treated 1.0 PEN membrane slides from Zeiss and stored at -80°C. Frozen sections were immediately submerged into ice cold RNase free 70% ethanol, stained in Vector® Hematoxylin QS for 30 sec, and then rinsed in RNase water, following by 95% ethanol and 100% ethanol. Slides were then air dried and returned to -80°C before laser capture. The surface ectoderm between the eye and brain was isolated using the Zeiss PALM into 0.5 mL tubes containing Qiagen RLT buffer. Three embryos were pooled together per sample and four wildtype and four knockout samples were collected for future analysis. RNA was isolated using a Qiagen RNeasy Plus Micro kit and assessed on an Agilent 2100 Bioanalyzer using Agilent RNA Pico Chips. All samples had a RIN above 7.0. cDNA synthesis and library preparation. cDNA was generated using Clontech's SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing according to manufacturer's recommendations. Sequencing libraries were prepared with 1 ng of cDNA using the Nextera XT DNA Library Prep Kit according to manufacturer's recommendations. The final product was then run on a Novex 4-12% TBE gel and DNA between 300-500 nt was selected. Samples were run on an Illumina HiSeq 2500 (UCSF Institute for Human Genetics Genomics Core). RNA-seq data analysis. Reads were mapped to the mouse GRCm38 genome using Tophat v.2.0.9 (Trapnell et al., 2009). Gene expression was measured from the mapped reads by using HT-seq-count (Anders et al., 2014) in intersection-strict mode, which counts the reads aligning to each annotated gene (gene set, Ensembl.org). Differentially expressed genes were called using the DESeq2 R package, (Anders and Huber, 2010) considering genes differentially expressed with FDR < 0.1. Only genes with normalized count values above 10 in all samples were evaluated. One WT samples (WT #1) was removed due to contamination of non-surface ectoderm tissues, as evident by decreased markers of Pax6, Krt5, and others that constant across all other samples. Please note that |
