The basic helix-loop-helix (bHLH) transcription factor Tal1 plays a critical role in the emergence of hematopoietic stem cells (HSCs) from their mesodermal precursors, repression of lymphoid fate specification in hematopoietic stem and progenitor cells (HSPC), development of megakaryocyte and erythroid progenitors, and is an oncogene that is aberrantly expressed in a majority of T acute lymphoblastic leukemias (T-ALL) and some myeloid leukemias. While there has been a great effort to understand the functions of Tal1, the underlying molecular mechanisms regulated by this transcription factor remain to be fully understood. My laboratory has been interested in the role of Tal1 in repressing lymphoid differentiation. While we have good evidence that Tal1 limits the frequency of lymphoid specified cells in the HSPC compartment, our current reagents do not allow us to determine whether Tal1 functions directly to suppress lymphoid gene priming in HSPCs or whether Tal1 is expressed, in and required for, development of a subset of HSCs that is biased away from lymphoid specification. To address this issue, we propose to create a mouse line in which the mCherry fluorescent protein is produced from the Tal1 gene so that Tal1 mRNA+ cells can be identified and isolated by flow cytometry or microscopy. This reporter mouse would be used to interrogate the heterogeneity in Tal1 expression in HSPCs and to determine whether Tal1 expression in HSCs is associated with myeloid biased HSCs or whether Tal1 is expressed in both lymphoid and myeloid biased HSCs. This reporter mouse will also be useful for studies on the initiation of aberrant Tal1 expression during leukemogenesis as well as in development of blood vessels, heart, and hematopoietic cells during embryonic development.
We propose to create a mouse line in which the mCherry fluorescent protein is produced from the Tal1 gene using CRISPR-Cas9-mediated cleavage and insertional mutagenesis. We will test the fidelity of reporter gene expression in multiple hematopoietic cell types by isolating subsets based on reporter expression and quantifiying Tal1 mRNA by quantitative real-time PCR. This Tal1 reporter line will be made available to scientific community for analyzing Tal1 expression patterns.
