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1.  Biomechanical force in blood development: extrinsic physical cues drive pro-hematopoietic signaling 
The hematopoietic system is dynamic during development and in adulthood, undergoing countless spatial and temporal transitions during the course of one’s life. Microenvironmental cues in the many unique hematopoietic niches differ, characterized by distinct soluble molecules, membrane-bound factors, and biophysical features that meet the changing needs of the blood system. Research from the last decade has revealed the importance of substrate elasticity and biomechanical force in determination of stem cell fate. Our understanding of the role of these factors in hematopoiesis is still relatively poor; however, the developmental origin of blood cells from the endothelium promts a model for comparison. Many endothelial mechanical sensors and second messenger systems may also determine hematopoietic stem cell fate, self renewal, and homing behaviors. Further, the intimate contact of hematopoietic cells with mechanosensitive cell types, including osteoblasts, endothelial cells, mesenchymal stem cells, and pericytes, places them in close proximity to paracrine signaling downstream of mechanical signals. The objective of this review is to present an overview of the sensors and intracellular signaling pathways activated by mechanical cues and highlight the role of mechanotransductive pathways in hematopoiesis.
PMCID: PMC3795909  PMID: 23850217
hematopoietic stem cells; hemogenic endothelium; biomechanical force; shear stress; mechanotransduction; cellular microenvironment
2.  Proliferation in the Absence of E2F1-3 
Developmental biology  2010;351(1):10.1016/j.ydbio.2010.12.025.
E2F transcription factors regulate the progression of the cell cycle by repression or transactivation of genes that encode cyclins, cyclin dependent kinases, checkpoint regulators, and replication proteins. Although some E2F functions are independent of the Retinoblastoma tumor suppressor (Rb) and related family members, p107 and p130, much of E2F-mediated repression of S phase entry is dependent upon Rb. We previously showed in cultured mouse embryonic fibroblasts that concomitant loss of three E2F activators with overlapping functions (E2F1, E2F2, and E2F3) triggered the p53-p21Cip1 response and caused cell cycle arrest. Here we report on a dramatic difference in the requirement for E2F during development and in cultured cells by showing that cell cycle entry occurs normally in E2f1-3 triply-deficient epithelial stem cells and progenitors of the developing lens. Sixteen days after birth, however, massive apoptosis in differentiating epithelium leads to a collapse of the entire eye. Prior to this collapse, we find that expression of cell cycle-regulated genes in E2F-deficient lenses is aberrantly high. In a second set of experiments, we demonstrate that E2F3 ablation alone does not cause abnormalities in lens development but rescues phenotypic defects caused by loss of Rb, a binding partner of E2F known to recruit histone deacetylases, SWI/SNF and CtBP-polycomb complexes, methyltransferases, and other co-repressors to gene promoters. Together, these data implicate E2F1-3 in mediating transcriptional repression by Rb during cell cycle exit and point to a critical role for their repressive functions in cell survival.
PMCID: PMC3868453  PMID: 21185283
Proliferation; Cell cycle; E2F; Rb; Lens; Repression; Cell survival; Transcription
3.  Atypical E2F Repressors and Activators Coordinate Placental Development 
Developmental cell  2012;22(4):849-862.
The evolutionarily ancient arm of the E2f family of transcription factors consisting of the two atypical members E2f7 and E2f8 is essential for murine embryonic development. However, the critical tissues, cellular processes and molecular pathways regulated by these two factors remain unknown. Using a series of fetal and placental lineage-specific cre mice we show that E2F7/E2F8 functions in extra-embryonic trophoblast lineages are both necessary and sufficient to carry fetuses to term. Expression profiling and biochemical approaches exposed the canonical E2F3a activator as a key family member that antagonizes E2F7/E2F8 functions. Remarkably, the concomitant loss of E2f3a normalized placental gene expression programs, corrected placental defects and fostered the survival of E2f7/E2f8 deficient embryos to birth. In summary, we identified a placental transcriptional network tightly coordinated by activation and repression through two distinct arms of the E2F family that is essential for extra-embryonic cell proliferation, placental development and fetal viability.
PMCID: PMC3483796  PMID: 22516201
4.  Biomechanical forces promote embryonic haematopoiesis 
Nature  2009;459(7250):1131-1135.
Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system1,2. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3–5), a master regulator of haematopoiesis, and give rise to haematopoietic cells4. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential6. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41+c-Kit+ haematopoietic progenitor cells7,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta–gonads–mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling8, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.
PMCID: PMC2782763  PMID: 19440194
5.  E2F1-3 Switch from Activators in Progenitor Cells to Repressors in Differentiating Cells 
Nature  2009;462(7275):930-934.
In the classic paradigm of mammalian cell cycle control, Rb functions to restrict cells from entering S phase by sequestering E2F activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase1, 2. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examine the effects of E2f1, E2f2 and E2f3 triple deficiency in murine ES cells, embryos and small intestines. We show that in normal dividing progenitor cells E2F1-3 function as transcriptional activators, but contrary to current dogma, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells they function in complex with Rb as repressors to silence E2F targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2F1-3 from repressors to activators, leading to the superactivation of E2F responsive targets and ectopic cell divisions, and loss of E2f1-3 completely suppressed these phenotypes. This work contextualizes the activator versus repressor functions of E2F1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles in vivo.
PMCID: PMC2806193  PMID: 20016602
Small intestine; cell cycle; E2F; retinoblastoma; tumor suppressor
6.  Bone marrow adipocytes as negative regulators of the hematopoietic microenvironment 
Nature  2009;460(7252):259-263.
Osteoblasts and endothelium constitute functional niches that support hematopoietic stem cells (HSC) in mammalian bone marrow (BM) 1,2,3 . Adult BM also contains adipocytes, whose numbers correlate inversely with the hematopoietic activity of the marrow. Fatty infiltration of hematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia 4. To explore whether adipocytes influence hematopoiesis or simply fill marrow space, we compared the hematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. By flow cytometry, colony forming activity, and competitive repopulation assay, HSCs and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 “fatless” mice, which are genetically incapable of forming adipocytes8, and in mice treated with the PPARγ inhibitor Bisphenol-A-DiGlycidyl-Ether (BADGE), which inhibits adipogenesis9, post-irradiation marrow engraftment is accelerated relative to wild type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone marrow microenvironment, and suggest that antagonizingmarrow adipogenesis may enhance hematopoietic recovery in clinical bone marrow transplantation.
PMCID: PMC2831539  PMID: 19516257
7.  Division and apoptosis in the E2f-deficient retina 
Nature  2009;462(7275):925.
E2fs 1-3, also known as activating E2fs, are viewed broadly as critical positive cell cycle regulators. They induce transcription and can drive cells out of quiescence. In flies and mammalian fibroblasts removing activating E2fs causes cell cycle arrest, suggesting an obligate proliferative role 1, 2. However, arrest is indirect as it is alleviated by removing the repressive E2f, dE2f2, in flies, or the tumor suppressor p53 in fibroblasts 3–5. Whether activating E2fs are required for division in vivo is thus an area of lively debate 6. Activating E2fs are also well known pro-apoptotic factors, a defense against oncogenesis 7. In some contexts E2f1 limits irradiation-induced apoptosis 8, 9, but in flies this occurs through repression of hid and the mammalian equivalent, Smac/Diablo is induced not repressed by E2f1 10, and in keratinoctyes it occurs indirectly through induction of DNA repair targets 11. Thus, a direct pro-survival function for activating E2fs in mammals has not been established. To address E2f1-3 function in vivo we focused on the mouse retina, a relatively simple CNS component that can be manipulated without compromising viability and has provided considerable insight into development and cancer 12–14. Here, we show that E2f1-3-deficient retinal progenitor cells or activated Muller glia divide. In the absence of activating E2fs, the Myc family drives proliferation. However, down-regulation of Sirt1, a p53 deacetylase, leads to hyperacetylation of p53 and cell death. Thus, activating E2fs are not universally required for mammalian cell division, but have an unexpected prosurvival role in development.
PMCID: PMC2813224  PMID: 20016601
E2f; Neurogenesis; p21Cip1; p57Kip2; Histone deacetylase; Sirtuin; p53; Resveratrol
8.  An Imaging Workflow for Characterizing Phenotypical Change in Large Histological Mouse Model Datasets 
Journal of biomedical informatics  2008;41(6):863-873.
This paper presents a workflow designed to quantitatively characterize the 3-D structural attributes of macroscopic tissue specimens acquired at a micron level resolution using light microscopy. The specific application is a study of the morphological change in a mouse placenta induced by knocking out the retinoblastoma gene.
This workflow includes four major components: (i) Serial-section image acquisition, (ii) image preprocessing, (iii) image analysis involving 2-D pair-wise registration, 2-D segmentation and 3-D reconstruction, and (iv) visualization and quantification of phenotyping parameters. Several new algorithms have been developed within each workflow component. The results confirm the hypotheses that (i) the volume of labyrinth tissue decreases in mutant mice with the retinoblastoma (Rb) gene knockout and (ii) there is more interdigitation at the surface between the labyrinth and spongiotrophoblast tissues in mutant placenta. Additional confidence stem from agreement in the 3-D visualization and the quantitative results generated.
The source code is available upon request.
PMCID: PMC2657595  PMID: 18502696
Light microscopy; histology staining; genetic phenotyping; mutation; morphometrics image analysis; image registration; segmentation; visualization; imaging workflow
9.  Rb-Mediated Neuronal Differentiation through Cell-Cycle–Independent Regulation of E2f3a 
PLoS Biology  2007;5(7):e179.
It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle–independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors.
Author Summary
The retinoblastoma protein (Rb), an important tumor suppressor, blocks division and death by inhibiting the E2f transcription factor family. In contrast, Rb is thought to promote differentiation by potentiating tissue-specific transcription factors, although differentiation defects in Rb null cells could be an indirect consequence of E2f-driven division and death. Here, we resolve different mechanisms by which Rb controls division, death, and differentiation in the retina. Removing E2f1 rescues aberrant division of differentiating Rb-deficient retinal neurons, as well as death in cells prone to apoptosis, and restores both normal differentiation and function of major cell types, such as photoreceptors. However, Rb-deficient starburst amacrine neurons differentiate abnormally even when E2f1 is removed, providing an unequivocal example of a direct role for Rb in neuronal differentiation. Rather than potentiating a cell-specific factor, Rb promotes starburst cell differentiation by inhibiting another E2f, E2f3a. This cell-cycle–independent activity broadens the importance of the Rb–E2f pathway, and suggests we should reassess its role in the differentiation of other cell types.
The retinoblastoma protein (Rb), a tumor suppressor, promotes the differentiation of starburst amacrine cells in the retina by inhibiting the transcription factor E2f3a, whereas it suppresses retinal cell division and death by inhibiting E2f1.
PMCID: PMC1914394  PMID: 17608565

Results 1-9 (9)