Elf5 is a principal cell lineage specific transcription factor in the kidney that contributes to Aqp2 and Avpr2 gene expression.

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.

The inositol phosphatase SHIP-1 is negatively regulated by Fli-1 and its loss accelerates leukemogenesis.

The activation of Fli-1, an Ets transcription factor, is the critical genetic event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. Fli-1 overexpression leads to erythropoietin-dependent erythroblast proliferation, enhanced survival, and inhibition of terminal differentiation, through activation of the Ras pathway. However, the mechanism by which Fli-1 activates this signal transduction pathway has yet to be identified. Down-regulation of the Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP-1) is associated with erythropoietin-stimulated erythroleukemic cells and correlates with increased proliferation of transformed cells. In this study, we have shown that F-MuLV-infected SHIP-1 knockout mice display accelerated erythroleukemia progression. In addition, RNA interference (RNAi)-mediated suppression of SHIP-1 in erythroleukemia cells activates the phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways, blocks erythroid differentiation, accelerates erythropoietin-induced proliferation, and leads to PI 3-K-dependent Fli-1 up-regulation. Chromatin immunoprecipitation and luciferase assays confirmed that Fli-1 binds directly to an Ets DNA binding site within the SHIP-1 promoter and suppresses SHIP-1 transcription. These data provide evidence to suggest that SHIP-1 is a direct Fli-1 target, SHIP-1 and Fli-1 regulate each other in a negative feedback loop, and the suppression of SHIP-1 by Fli-1 plays an important role in the transformation of erythroid progenitors by F-MuLV.

Increasing Students' Publication Productivity: Could Launching a University Scientific Journal be a Catalyst?

Our institution established an online medical journal to promote publication opportunities and to foster a culture of scholarship. In two years of activity, there was an increase in the proportion of students reporting authorship of peer-reviewed publications at our institution suggesting an increase in students' scholarly interest and output.

Online Faculty Development for Implementation and Use of Student Portfolios.

INTRODUCTION: Over the past decade, portfolios have gained popularity in medical education as tools to evaluate and provide feedback about learning and completion of professionally authentic tasks. Though faculty development has been noted to be key for successful portfolios, there are few available resources. As part of an interinstitutional collaborative project, we have developed online faculty development modules that provide pedagogical information about portfolios, practical advice, and resources from the available literature. METHODS: The materials associated with this publication include downloadable modules, which take approximately 45 minutes to complete and can be paused at any time, and sample questions to facilitate small-group discussion with faculty either in the planning stage of portfolios or as part of program evaluation of an institution's portfolios. RESULTS: A survey taken by faculty from four medical schools after completion of the modules showed that they were well received, with 41% of participants stating that they were very knowledgeable after undertaking the modules compared to 11% before undertaking the modules. Faculty reported increased interest in the topic and increased confidence in their ability to undertake planning for development of portfolios at their institution and considered using the modules as a mandatory curriculum for portfolio coaches at their institutions. DISCUSSION: We suggest that these modules be used for individual self-development, as part of faculty development sessions for portfolio coaches and mentors, or to provide faculty with background information about portfolios during the planning phase of portfolios at an institution.

Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X)), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-)). Timed pregnancy experiments revealed that Jak2(K915X/K915X) and Jak2(-/-) displayed embryonic lethality; however, Jak2(K915X/K915X) embryos were viable an additional two days compared to Jak2(-/-) embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

Cytopenia induction by 5-fluorouracil identifies thrombopoietic mutants in sensitized ENU mutagenesis screens.

The ability of random mutagenesis techniques to annotate the mammalian genome can be hampered due to genetic redundancy and compensatory pathways that mask heterozygous mutations under homeostatic conditions. The objective of this study was to devise a pharmacologically sensitized screen using the chemotherapeutic drug, 5-fluorouracil (5FU), to induce cytopenia. 5FU dose was optimized in the 129/SvImJ, C57BL/6J, BALB/cJ, and C3H/HeJ strains of laboratory mice. N-ethyl-N-nitrosourea (ENU) mutagenesis was performed on 129/SvImJ males and phenotypic variants were identified by backcrossing on to the C57BL/6J background. G1 animals were challenged with 100 µg/g 5FU and phenodeviants with altered platelet recovery were monitored. Of 546 G1 animals tested, 15 phenodeviants were identified that displayed increased baseline platelet number, a platelet overshoot, or delayed platelet recovery, thereby demonstrating the utility of this approach for uncovering mutations in megakaryocyte and platelet development. Four G1 mice were selected for further analysis. The phenotypes were heritable in all four strains and genetic mapping identified a chromosome location in two of the three G2 lines tested. In conclusion, our group has developed a sensitized random mutagenesis screen utilizing 5FU and has shown that the strain combination of 129/SvImJ × C57BL/6J is robust for identification of founder lines with defects in megakaryocyte and platelet development.

Src homology 2 domain-containing inositol 5-phosphatase 1 deficiency leads to a spontaneous allergic inflammation in the murine lung.

BACKGROUND: Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) controls the intracellular level of the phosphoinositide 3-kinase product phosphotidylinositol-3,4,5-trisphosphate and functions as a negative regulator of cytokine and immune receptor signaling. Emerging evidence suggests that the phosphoinositide 3-kinase pathway might be involved in allergic inflammation in the lung. However, the functional relevance of SHIP-1 in the T(H)2 activation pathway has not been established. SHIP-1(-/-) mice have spontaneous myeloproliferative inflammation in the lung, the nature of which has not been elucidated. We hypothesized that SHIP-1 plays an important role as a regulator in pulmonary allergic inflammation and in maintaining lung homeostasis. OBJECTIVE: To test our hypothesis, we characterized the pulmonary phenotype of SHIP-1(-/-) mice. RESULTS: Analyses of lung histopathology and bronchoalveolar lavage cellularity revealed that the majority of SHIP-1(-/-) mice had progressive and severe pulmonary inflammation of macrophages, lymphocytes, neutrophils, and eosinophils; mucous hyperplasia; airway epithelial hypertrophy; and subepithelial fibrosis. These pathologic changes were accompanied by exaggerated production of T(H)2 cytokines and chemokines, including IL-4, IL-13, eotaxin, and monocyte chemoattractant protein 1, in the lung. Furthermore, the number of mast cells significantly increased, and many of these cells were undergoing degranulation, which was correlated with increased content and spontaneous release of histamine in the lung tissue of SHIP-1(-/-) mice. CONCLUSION: These findings provide strong evidence that mice lacking SHIP-1 have an allergic inflammation in the lung, suggesting that SHIP-1 plays an important role in regulating the T(H)2 signaling pathway and in maintaining lung homeostasis. CLINICAL IMPLICATIONS: SHIP-1 as a regulator might be a potential therapeutic target for controlling allergic inflammation in diseases such as asthma.

A novel role for STAT1 in regulating murine erythropoiesis: deletion of STAT1 results in overall reduction of erythroid progenitors and alters their distribution.

Erythropoietin (EPO) activates many distinct signal transduction cascades on engagement of its receptor. Deletion of the EPO, EPO receptor (EPO-R), or JAK2 genes in mice results in embryonic lethality due to a fatal anemia. EPO activates signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5a/b transcription factors in erythroid cell lines. Studies have focused on STAT5 as the primary target of EPO-dependent JAK2 activation. However, STAT5a/b(-/-) mice are viable, displaying a nonfatal anemia during embryogenesis, and delayed differentiation in adult erythropoiesis. Importantly, EPO-R cytoplasmic tyrosines are dispensable for viability in vivo. Interestingly, no cytoplasmic tyrosines are required for phosphorylation of STAT1. This led us to examine whether STAT1-deficient mice have altered erythropoiesis. A shift in erythropoiesis was observed in STAT1(-/-) mice, with reduced bone marrow-derived erythroid colony-forming units (CFU-Es) and a compensatory increase in splenic burst-forming units (BFU-Es) and CFU-Es. Both types of splenic-derived cells displayed EPO hyperresponsiveness. A 1.6-fold reduction in total CFU-Es was observed in STAT1-deficient mice, whereas total BFU-Es were comparable. Flow cytometry of STAT1-deficient erythroid cells revealed a less differentiated phenotype, associated with increased apoptosis of early erythroblasts. STAT1-deficient erythroblasts from phenylhydrazine-primed mice displayed enhanced phosphorylation of STAT5a/b, Erk1/2, and protein kinase B (PKB)/Akt. These results illustrate that STAT1 plays an important role in the regulation of erythropoiesis.