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.

Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis.

The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic ß-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 ß-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.

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, ß-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.

Identification of erythroid-enriched gene expression in the mouse embryonic yolk sac using microdissected cells.

Little is known about the genes that control the embryonic erythroid program. Laser capture microdissection was used to isolate primitive erythroid precursors and epithelial cells from frozen sections of the embryonic day 9.5 yolk sac. The RNA samples were amplified and labeled for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. Ninety-one genes are expressed significantly higher in erythroid than in epithelial cells. Ingenuity pathway analysis indicates that many of these erythroid-enriched genes cluster in highly significant biological networks. One of these networks contains RBTN2/LMO2, SCL/TAL1, and EKLF/KLF1, three of the very few genes required for primitive erythropoiesis. Quantitative real-time polymerase chain reaction was used to verify that platelet factor 4, reelin, thrombospondin-1, and muscleblind-like 1 mRNA is erythroid-enriched. These genes have established roles in development or differentiation in other systems, and are, therefore, good candidates for regulating primitive erythropoiesis. These results provide a catalog of genes expressed during primitive erythropoiesis.

EKLF and KLF2 have compensatory roles in embryonic beta-globin gene expression and primitive erythropoiesis.

The Krüppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Krüppel-like factor (EKLF or KLF1) regulates adult beta-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey- and betah1-, but not adult betaglobin, gene expression in mice. Both EKLF and KLF2 play roles in primitive erythroid cell development. To investigate potential interactions between these genes, EKLF/KLF2 double-mutant embryos were analyzed. EKLF(-/-)KLF2(-/-) mice appear anemic at embryonic day 10.5 (E10.5) and die before E11.5, whereas single-knockout EKLF(-/-) or KLF2(-/-) embryos are grossly normal at E10.5 and die later than EKLF(-/-)KLF2(-/-) embryos. At E10.5, Ey- and betah1-globin mRNA is greatly reduced in EKLF(-/-)KLF2(-/-), compared with EKLF(-/-) or KLF2(-/-) embryos, consistent with the observed anemia. Light and electron microscopic analyses of E9.5 EKLF(-/-)KLF2(-/-) yolk sacs, and cytospins, indicate that erythroid and endothelial cells are morphologically more abnormal than in either single knockout. EKLF(-/-)KLF2(-/-) erythroid cells are markedly irregularly shaped, suggesting membrane abnormalities. EKLF and KLF2 may have coordinate roles in a common progenitor to erythroid and endothelial cells. The data indicate that EKLF and KLF2 have redundant functions in embryonic beta-like globin gene expression, primitive erythropoiesis, and endothelial development.

Identification, characterization, and expression pattern of the chicken EKLF gene.

EKLF/KLF1 was the first of the Krüppel-like factors (KLFs) to be identified in mammals and plays an important role in primitive and definitive erythropoiesis. Here, we identify and characterize EKLF in the chicken (cEKLF). The predicted amino acid sequence of the zinc finger region of cEKLF is at least 87.7% similar to mammalian EKLF proteins and is 98.8% and 95% similar to the EKLF orthologues in Xenopus and zebrafish, respectively. During early embryonic development, cEKLF expression is seen in the posterior primitive streak, which gives rise to hematopoietic cells, and then in the blood islands and in circulating blood cells. cEKLF mRNA is expressed in blood cells but not in brain later in chicken embryonic development. cEKLF mRNA is increased in definitive compared with primitive erythropoiesis. The conserved sequence and expression pattern of cEKLF suggests that its function is similar to its orthologues in mammals, Xenopus, and zebrafish.

Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.

Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.

A functional screen for Krüppel-like factors that regulate the human gamma-globin gene through the CACCC promoter element.

Krüppel-like factors (KLFs) have been systematically screened as potential candidates to regulate human gamma-globin gene expression through its CACCC element. Initially, 21 human proteins that have close sequence similarity to EKLF/KLF1, a known regulator of the human beta-globin gene, were identified. The phylogenetic relationship of these 22 KLF/Sp1 proteins was determined. KLF2/LKLF, KLF3/BKLF, KLF4/GKLF, KLF5/IKLF, KLF8/BKLF3, KLF11/FKLF, KLF12/AP-2rep and KLF13/FKLF2 were chosen for functional screening. Semi-quantitative RT-PCR demonstrated that all eight of these candidates are present in human erythroid cell lines, and that the expression of the KLF2, 4, 5 and 12 mRNAs changed significantly upon erythroid differentiation. Each of the eight KLF mRNAs is expressed in mouse erythroid tissues, throughout development. UV cross-linking assays suggest that multiple erythroid proteins from human cell lines and chicken primary cells interact with the gamma-globin CACCC element. In co-transfection assays in K562 cells, it was demonstrated that KLF2, 5 and 13 positively regulate, and KLF8 negatively regulates, the gamma-globin gene through the CACCC promoter element. The data collectively suggest that multiple KLFs may participate in the regulation of gamma-globin gene expression and that KLF2, 5, 8 and 13 are prime candidates for further study.

Evolutionary conservation of KLF transcription factors and functional conservation of human gamma-globin gene regulation in chicken.

The Krüppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins with homology to Drosophila Krüppel. KLFs can bind to CACCC elements, which are important in controlling developmental programs. The CACCC promoter element is critical for the developmental regulation of the human gamma-globin gene. In the present study, chicken homologues of the human KLF2, 3, 4, 5, 9, 11, 12, 13, and 15 genes were identified. Phylogenetic analysis confirms that these genes are more closely related to their human homologues than they are to other chicken KLFs. This work also represents the first systematic study of the expression patterns of KLFs during erythroid development. In addition, transient transfections of human globin constructs into 5-day (primitive) chicken red blood cells show that human gamma-globin expression is regulated via its CACCC promoter element. This indicates that a CACCC-binding factor(s) important for gamma-globin expression functions in 5-day chicken red cells.