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1.  Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays 
In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.
doi:10.1007/978-1-60327-292-6_4
PMCID: PMC4096662  PMID: 24318813
Laser capture microdissection; RNA; Microarray; Infrared laser
2.  The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis 
Background
Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied.
Results
Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos.
Conclusions
This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.
doi:10.1186/1471-213X-13-40
PMCID: PMC4222490  PMID: 24261709
KLF2; KLF4; Gene interactions; Embryonic vascular integrity; Endothelial cell development
3.  Krüppel-Like Factor 2 Is Required for Normal Mouse Cardiac Development 
PLoS ONE  2013;8(2):e54891.
Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2−/− embryos die earlier, by E11.5. E9.5 FVB/N KLF2−/− hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2−/− AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2−/− hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2−/− AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2−/− embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2−/− hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2−/− embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF2−/− heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important cardiovascular genes.
doi:10.1371/journal.pone.0054891
PMCID: PMC3573061  PMID: 23457456
4.  Krüppel-Like Factor 1 (KLF1), KLF2, and Myc Control a Regulatory Network Essential for Embryonic Erythropoiesis 
Molecular and Cellular Biology  2012;32(13):2628-2644.
The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic β-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1−/− KLF2−/− double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1−/−, and KLF1−/− KLF2−/− mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1−/− KLF2−/− embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and β-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.
doi:10.1128/MCB.00104-12
PMCID: PMC3434496  PMID: 22566683
5.  Krüppel-like Factor 2 Regulated Gene Expression in Mouse Embryonic Yolk Sac Erythroid Cells 
KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. In KLF2-/- embryos, β-like globin gene expression is reduced, and E10.5 erythroid cells exhibit abnormal morphology. In this study, other genes regulated by KLF2 were identified by comparing E9.5 KLF2-/- and wild-type (WT) yolk sac erythroid precursor cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression between KLF2-/- and WT; eighty-nine of these are downregulated in KLF2-/-. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. The SOX2 gene, encoding a pluripotency factor, is regulated by KLF2 in both ES and embryonic erythroid cells. Previous work had identified genes with erythroid-enriched expression in the yolk sac. The erythroid-enriched genes reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT, and are therefore candidates for controlling primitive erythropoiesis. Each of these genes contains a putative KLF2 binding site(s) in its promoter and/or an intron. Reelin has an established role in neuronal development. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter in erythroid cells, validating this approach to identify KLF2 target genes.
doi:10.1016/j.bcmd.2011.03.002
PMCID: PMC3150518  PMID: 21530336
yolk sac; embryonic erythropoiesis; KLF2; expression profiling; laser capture microdissection
6.  Expression patterns of astrocyte elevated gene-1 (AEG-1) during development of the mouse embryo 
Gene expression patterns : GEP  2010;10(7-8):361-367.
Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.
doi:10.1016/j.gep.2010.08.004
PMCID: PMC3165053  PMID: 20736086
AEG-1; development; mouse embryo; cell proliferation; cancer
7.  Erythropoietin-Induced Phosphorylation/Degradation of BIM Contributes to Survival of Erythroid Cells 
Experimental hematology  2008;37(2):151-158.
Objective
A pro-apoptotic BH3-only protein BIM (BCL-2 interacting mediator of cell death) can link cytokine receptor signaling with the apoptotic machinery in hematopoietic cells. We investigated here the role of BIM in erythropoietin (Epo)-mediated survival in erythroid cells.
Methods
We down-regulated BIM in Epo-dependent HCD57 erythroid cells with shRNA, and used Real-time PCR, Western blots, and flow cytometry to characterize BIM expression and apoptosis. Hematologic analyses of BIM-deficient (Bim−/−) mice were conducted.
Results
BIM expression increases in primary murine erythroid cells and HCD57 cells deprived of Epo. Whereas Bim mRNA increased less than 2-fold, BIM protein increased more than 10-fold after Epo withdrawal, suggesting post-transcriptional regulation of BIM. Epo treatment resulted in rapid phosphorylation of BIM at Serine 65 and phosphorylation correlated with degradation of BIM. Inhibition of ERK (extracellular signal-regulated kinase) by a MEK/ERK inhibitor, U0126, blocked both phosphorylation and degradation of BIM, resulting in apoptosis. Treatment with a proteasome inhibitor, MG-132, also blocked degradation of phosphorylated BIM. Down-regulation of BIM with the shRNA resulted in HCD57 cells more resistant to apoptosis induced by either Epo withdrawal or ERK inhibition. Although we observed no significant changes in the number of erythrocytes or reticulocytes in the circulation of Bim−/− mice, erythroid progenitors from bone marrow in Bim−/− mice were reduced in number and more resistant to apoptosis induced by U0126 MEK/ERK inhibitor.
Conclusion
Epo protects erythroid cells from apoptosis in part through ERK-mediated phosphorylation followed by proteasomal degradation of BIM.
doi:10.1016/j.exphem.2008.10.008
PMCID: PMC2656114  PMID: 19100675
erythroid; erythropoietin; apoptosis; BIM; ERK

Results 1-7 (7)