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Cover Letter Re: A New Method of Mining Developmentally Regulated Genes Identifies a Branchpoint Between B and T Cell Development Debashis Sahoo*, Matthew A. Inlay*, Jun Seita, Holger Karsunky, Thomas Serwold, Sylvia K. Plevritis, Irving L. Weissman, Deepta Bhattacharya, and David L. Dill * These authors contributed equally to this work Dear Editor, Please find accompanying this letter a copy of our manuscript entitled, "A New Method of Mining Developmentally Regulated Genes Identifies a Branchpoint Between B and T Cell Development," that we submit for consideration for publication as an article in Nature. In this manuscript we present a novel computational method, MiDReG (Mining Developmentally Regulated Genes), which enables the identification of developmentally regulated genes using publicly available microarray data. Differentiation from precursor cells to mature progeny typically proceeds through developmental intermediates, where a network of gene expression changes gradually establishes the identity of the mature lineage and extinguishes the potential for alternative lineages. Understanding the molecular mechanisms of lineage commitment critically depends upon the ability to identify and isolate cells at intermediate stages of development; however, identification of these intermediates is challenging. MiDReG enables logical predictions of genes that might define such intermediates. MiDReG bases its predictions on if-then gene expression relationships (Boolean implications) that hold consistently over thousands of microarrays from hundreds of different laboratories, mined using a technique we recently developed. Here, we demonstrate the utility of MiDReG for identifying new genes functionally important to B cell biology. In addition, we applied MiDReG to an existing question in lymphoid development, and discover a surface marker, Ly6d, that identifies the branchpoint between B cell and T cell development. The major specific findings of this study are as follows: 1. MiDReG was validated using a well-characterized system, B cell development. MiDReG predicted 19 genes to be upregulated during the transition from KIT+ hematopoietic stem/progenitors to CD19+ B cells. By quantitative RT-PCR, we show that 71% of analyzed genes were upregulated during this transition. We expanded our list of predicted B cell genes to 62 through the combination of multiple genes. Literature investigation revealed that 36 of these genes have been knocked out in mouse, of which 19 show specific defects in B cell function and differentiation. Therefore, MiDReG reliably predicts novel genes that are functionally related in B cell development. 2. MiDReG identified Ly6d, a surface marker which separates bone marrow-derived common lymphoid progenitors (CLP) into two distinct fractions. Ly6d- CLP are unbiased for either B or T cell differentiation, but are lymphoid committed, whereas Ly6d+ CLP are almost completely B cell committed. Therefore, Ly6d expression marks the first step in B cell specification, and is the earliest marker for B cell development identified to date. The Ly6d- CLP lies at a developmental checkpoint immediately upstream of lineage commitment to the B, T, NK, or DC cell fates, identifying it as a uniquely important intermediate in hematopoiesis. We feel that our manuscript will be of great general interest to the readership of Nature. MiDReG is a powerful tool to identify novel genes and cellular stages in development, and is applicable to any developmental pathway where some knowledge of the genes expressed at the beginning and end stages are known. Importantly, in our analysis no intermediate B cell populations were represented in the microarrays. To identify these genes using a traditional array based approach would have required careful sorting of each of the intermediate populations and thousands of dollars in microarrays. MiDReG uses microarray data that has already been generated and is freely available to predict genes that change during developmental progressions. Furthermore, we have used MiDReG to address an important biological question that had not previously been answered using traditional approaches. As a result, we have isolated the population making the T vs. B cell fate decision, which will now allow the careful dissection of the molecular mechanisms involved in that decision. We are aware that a controversy exists as to the nature of the thymic seeding population, and that many groups claim that a myeloid/lymphoid progenitor (LMPP) is that population. Just this month at the AAI conference, nearly every talk that presented a hematopoietic tree had CLP producing only B cells. Our data clearly demonstrate that Ly6d- CLP produce T cells in the thymus earlier than the upstream LMPP. While we feel that the introduction of Ly6d will improve labs’ abilities to isolate this population, and is therefore an important contribution toward resolving this field, the major point of this paper is to introduce MiDReG, a method which is applicable to all of developmental biology. Ideally, this manuscript should be critiqued by reviewers who are familiar with both computational theory and developmental biology, rather than by those only interested in this manuscript’s relevance to the CLP/MPP controversy. As potential reviewers, we recommend Andrea Califano (Columbia University Medical Center), Ihor Lemischka (Mount Sinai School of Medicine), Hans-Reimer Rodewald (Ulm Institute for Immunology) and Cornelis Murre (University of California, San Diego). We respectfully request to exclude David Allman and his collaborators Avinash Bhandoola and Richard Hardy, due to potential conflicts of interest. Because this manuscript spans both Computational Biology and Developmental Immunology, we would like to have two corresponding authors listed: David Dill (Computational Biology) and Matthew Inlay (Immunology). Thank you for your consideration, David L. Dill and Irving L. Weissman