Functions And Mechanisms Of LIM Homeodomain Transcription Factor Isl1in The Mouse Pyloric Development

A recent report has shown that Isll is expressed in the mesenchyme and epithelium of the embryonic stomach in mice. However, it is unclear that regarding its spatiotemporal expression, functions and the related mechanisms in the pyloric development. First, we analyze Isl1expression in different developmental stages of the mouse pylorus by in situ hybridization (ISH), real-time quantitative PCR (RT-qPCR) and immunohistochemistry (IHC). Secondly, we identified the Isll biological functions in pyloric development utilizing an inducible Isl1knockout mouse model. Finally, we further study the signaling pathways and molecular mechanisms of Isl1in the pyloric development using chromatin immunoprecipitation (ChIP), dual luciferase vector and electrophoretic mobility shift assays (EMSA) methods. The most important results are as follows:1. RT-qPCR results showed that Isll mRNA reached the highest level at E13.5, followed by a sharp decline at E14.5and had no significant changes into adulthood. ISH and IHC results demonstrated that Isl1was localized to the smooth muscle layer of the pylorus.2. Isll and α-SMA double immunofluorescence results showed that the proportion of Isll positive cells expressing α-SMA gradually increased. Meantime, Isll was expressed in human pylorus with the hypertrophic pyloric stenosis. These results suggested that Isll may participate in the muscle formation of pylorus.3. Tamoxifen inducible Isll knockout mice exhibited the deletion of exon4. Western blot and immunofluorescence results showed that Isl1protein levels in embryonic stomach of Isl1MCM/Del mice were significantly lower than those in wild type (Isl1F/+) mice. These collective data showed that Isl1expression was efficiently mutated in the IsllMCM/Del mice.4. Isl1deficiency had no significant effect on epithelium and nerve development, but led to nearly complete absence of the dorsal pyloric outer longitudinal muscle (OLM) layer.5. RT-qPCR results showed that Isll knockout down-regulated Nkx2.5, Gata3and Gremlin mRNA expression in the embryonic stomach both at E14.5and E18.5. Whole-mount ISH results showed that the ISH signals of aforementioned genes were much weaker in the Isl1MCM/Del mouse stomach than in Isl1F/+at E14.5. Gata3mRNA levels decreased about70%at both examined stages.6. Double immunofluorescence results showed that Isll and Gata3were co-expressed in the pyloric smooth muscle cells. In addition, Gata3P1and P6regions were occupied by Isll protein through ChIP.7. Dual-luciferase reporter assay showed that Isll enhanced the activity of the Gato3-P1-WT (wild type, WT) luciferase reporter, but Isl1did not affect luciferase activities of Gata3-P1-MT (mutant type, MT), Gata3-P6-WT, Gata3-P6-MT and pGL3.0-basic.8. Gata3-P1promoter region includes three putative ATTA binding sites, and EMSA assay showed that Isll efficiently bound to oligonucleotides representing number1and number3sites. These collective data strongly showed that Isl1was a direct regulator of Gata3transcription. Taken together, LIM homeodomain (LIM-HD) transcription factor Isl1was strongly expressed in the smooth muscle cells of pylorus, and involved in regulating dorsal pyloric OLM development via directly targeting Gata3. In addition, these findings suggested that Isl1may be related to hypertrophic pyloric stenosis.