Embryonic Intra-Aortic Clusters Undergo Myeloid Differentiation Mediated by Mesonephros-Derived CSF1 in Mouse
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The aorta-gonad-mesonephros (AGM) region contains intra-aortic clusters (IACs) thought to have acquired hematopoietic stem cell (HSC) potential in vertebrate embryos. To assess extrinsic regulation of IACs in the AGM region, we employed mouse embryos harboring a Sall1-GFP reporter gene, which allows identification of mesonephros cells based on GFP expression. Analysis of AGM region tissue sections confirmed mesonephros GFP expression. Mesonephric cells sorted at E10.5 expressed mRNA encoding Csf1, a hematopoietic cytokine, and corresponding protein, based on real-time PCR and immunocytochemistry, respectively. Further analysis indicated that some IACs express the CSF1 receptor, CSF1R. Expression of Cebpa and Irf8 mRNAs was higher in CSF1R-positive IACs, whereas that of Cebpε and Gfi1 mRNAs was lower relative to CSF1R-negative IACs, suggesting that CSF1/CSF1R signaling functions in IAC myeloid differentiation by modulating expression of these transcription factors. Colony formation assays using CSF1R-positive IACs revealed increased numbers of myeloid colonies in the presence of CSF1. Analysis using an intra-cellular signaling array indicated the greatest fold increase of Cleaved Caspase-3 in AGM cells in the presence of CSF1. Immunohistochemistry revealed that Cleaved Caspase-3 is primarily expressed in IACs in the AGM region, and incubation of IACs with CSF1 up-regulated Cleaved Caspase-3. Overall, our findings suggest that CSF1 secreted from mesonephros accelerates IAC myeloid differentiation in the AGM region, possibly via Caspase-3 cleavage.
KeywordsIntra-aortic clusters Hematopoietic stem cells AGM region Mesonephros CSF1 Myeloid differentiation
This work was supported by a Grant-in-Aid for Exploratory Research by the Project for Realization of Regenerative Medicine, of the Ministry of Education, Culture, Sports, Science and Technology; and by a Bilateral grant to promote scientific exchange between Germany and Japan from the Japan Society for the Promotion of Science. We thank Dr. Elise Lamar for critical reading of our manuscript; Ms. Chiyo Yanagi-Mizuochi for technical support.
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Conflict of Interest
The authors declare no potential conflicts of interest.
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