By seven days of gestation, the yolk sac of the mouse has a sheet of mesoderm adjacent to the basement membrane separating it from the endodermal epithelium. Localized proliferations of this mesoderm produces thickened cellular regions which transform into the angioblastic cords; all of these developmental cells are attached by tight junctions and desmosomes. By eight and onehalf days, lumina appear within the angioblastic cords; the peripheral cells become attenuated and form endothelial cells which will line the primitive vessels while the more central cells become the primitive erythroblasts of the blood island.The process of vasculogenesis and lumenization occurs between eight and onehalf and nine days of gestation and has been correlated with the reduction of cellular junctions between angioblasts and fixed primitive erythroblasts, a loss of the visceral basement membrane and the formation of wide intercellular channels between endodermal epithelial cells. The primitive erythroblasts comprising the blood islands have abundant polysomes, sparse rough endoplasmic reticulum and possess coated vesicles and ferritin aggregates in their cytoplasm and coated invaginations of their plasma membrane. By nine days of gestation, the primitive erythroblasts lose their attachments and become free in the vitelline vessels. Mitochondria of the primitive and free erythroblasts are slightly enlarged and have lighter matrices than angioblasts and mesodermal cells. By 10 to 11 days of gestation, as differentiation proceeds, coated vesicles and invaginations become more numerous and the developing erythroblasts gradually decrease in both cell and nuclear size. Concomitant with these changes is the decrease in the number and size of the mitochondria, a decrease in polysomal numbers and an increase in hemoglobin and cytoplasmic density.The inter-and intracellular events that occur during the formation of the primitive blood vessels and erythrocytic precursors of the yolk sac have not been thoroughly documented from an ultrastructural standpoint. The developmental stages in the process of hemopoiesis occur rapidly with the undifferentiated cells undergoing irreversible specialization along distinct pathways. Submicroscopic analysis has the dual potential of determining the earliest morphological features which signal the initial steps of differentiation and illuminating the changes in structural and functional relationships that occur during subsequent maturation and specialization. Previous studies have dealt with such features as the cellular versus syncytial nature of the primitive angioblastic cords, how ANAT. REC., 170: 199-224. vascular lumen form and the developmental pathway and structural features of erythrocyte differentiation and maturation (Bloom, '38; Danchakoff, '16a,b; Haar, '70; Jones, '64; Kovach et al., '67; Maximow, '09, '24; Sorenson, '61). It is the purpose of this correlated phase and electron microscopic study to expand and clarify the early developmental processes involved in erythropoiesis and vas...

The Krü ppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krü ppel-like factor (EKLF or KLF1) positively regulates the ␤-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2 ؊/؊ mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the ␤-like globin genes in KLF2 knockout mice. Our results show that KLF2 ؊/؊ mice have a significant reduction of murine embryonic Ey-and ␤h1-globin but not -globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult ␤ maj -and ␤ min -globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic ⑀-globin gene but not the adult ␤-globin gene, suggesting that this developmentalstage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2 ؊ IntroductionHematopoiesis represents a complex differentiation pathway involving many transcription factors and growth factors that interact in a concerted fashion during mammalian development. 1 Transcription factors often exist as multigene families of structurally and functionally related members that are involved in the different stages of development of a particular cell lineage. For example, members of the GATA family of transcription factors are involved in both primitive and definitive erythropoiesis. 2-4 Different phenotypic features seen after ablation of either the GATA1 or the GATA2 gene in mice clearly demonstrate that these factors have different but overlapping functions. The Krüppel-like factors (KLFs) are a family of DNA-binding proteins named after the Drosophila Krüppel protein. KLFs have 3 C2/H2 zinc finger domains and share conserved residues located primarily within these zinc fingers. 5,6 Several of the KLFs are expressed in erythroid cells starting early in development. Erythroid Krüppel-like factor (EKLF or KLF1) was the first of 16 KLFs to be identified. EKLF Ϫ/Ϫ mice develop fatal anemia during fetal liver erythropoiesis. 7,8 EKLF is responsible for positively regulating the adult ␤-globin gene, but it is not required for embryonic/fetal globin gene expression. [9][10][11] Other KLF family members may be involved in the developmental control of the embryonic and fetal globin genes. A few of the KLFs, namely KLF2 and KLF5, 12 and KLF11 and KLF13,13,14 have been shown to activate the fetal ␥-globin gene in transient transfection assays in human erythroleukemia cell lines. So far none of these studies have been replicated in vivo. In a recent study, KLF11 (fetal Krüppel like factor, FKLF1)-null mice were found to be fertile, with normal hematopoiesis at all stages of development. There was no effe...

Mouse fetal liver was studied ultrastructurally to identify and characterize the developing hepatic parenchyma or prehepatocyte which may be responsible for producing the liver hemopoietic environment. It was observed that as the liver develops, there is close association of endodermal and mesenchymal cells in the region of the septum transversum. Numerous intercellular adhesions were observed between endodermal cells and mesenchymal cells. Twenty-four after endodermal and mesenchymal cells first intermingle, the liver extravascular space consisted of spherical hemopoietic cells dispersed among a heterogenous population of dark and light cells. The reticulum of prehepatocytes formed a three-dimensional cellular network which structurally supported the hemopoietic cells residing in the liver. By 12 days of gestation, prehepatocytes were a homogenous population of dark, stellate cells joined together by numerous intercellular adhesions. Broad areas of intercellular association were noted between processes and prehepatocytes and hemopoietic cells; however, no intercellular junctions between these two disparate cell populations were observed at this or any stage in development. Characteristics reflecting a cell population capable of synthesis and secretion of proteinaceous substances, namely, dilated Golgi apparati, increased numbers of polyribosomes and profiles of rough endoplasmic reticulum (RER), two types of vacuoles and/or vesicles, and intercellular microvillus-lined spaces, were observed in the prehepatocytes between 12 and 17 days gestation. By day 17 of gestation, glycogen accumulation, biliary channel development, appearance of a subendothelial microvillus surface, nuclear shape and chromatin pattern, and arrangement of cytoplasmic organelles reflected the maturation of prehepatocytes into hepatocytes, the adult liver parenchyma.

BackgroundKrüppel-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.ResultsEndothelial 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.ConclusionsThis 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.

The morphology of early postimplantation mouse egg cylinders was studied using light and electron microscopy. Implantation sites at seven, seven and one-half and eight days of gestation were dissected from the myometrium and whole implants, including both decidua and egg cylinders were processed for electron microscopy. Pre-primitive streak egg cylinders were composed of two germ layers, a tall columnar ectoderm and an outer visceral endodermal layer. Ectodermal cells demonstrated large oval nuclei and an organelle sparse cytoplasm except for many free polyribosomes. The visceral endodermal layer was composed of two cell populations. One visceral endodermal cell type observed was tall columnar in shape and appeared absorptive as demonstrated by many microvilli, pinocytotic profiles and lysosomal granules. This population was confined to extraembryonic regions of the egg cylinder. The second visceral endodermal cell type, squamous in shape, evidenced only a few microvilli, pinocytotic profiles and lysosomal granules. This population was confined to the embryonic region of the egg cylinder. Concurrent with the formation of the primitive streak an increased number of cellular junctions and nuclear pores became evident in the ectoderm. Mesodermal cells were large and stellate-shaped exhibiting many filapodia which made contact with adjacent mesodermal elements. Later the cephalic region of the primitive streak proliferated resulting in the migration of wedge-shaped mass of cells, the head process. At the most ventral extremity of the post-primitive streak egg cylinder the cells of the head process became intimately associated with the ectoderm by areas of focal contact and gap junctions.

The Krü ppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Krü ppel-like factor (EKLF or KLF1) regulates adult ␤-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey-and ␤h1-, but not adult ␤-globin, 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 ␤h1-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 IntroductionKrüppel-Like factors (KLFs) are a family of DNA-binding proteins with sequence homology to the Drosophila transcription factor, Krüppel. KLFs have 3 C2/H2 zinc finger domains and share conserved residues located primarily within these domains. 1,2 Erythroid Krüppel-Like factor (EKLF or KLF1) was the first of 17 KLFs to be identified in mouse and man. 3 It is expressed specifically in erythroid cells and positively regulates the adult ␤-globin gene. 3,4 EKLF Ϫ/Ϫ mice develop fatal anemia during definitive (fetal liver) erythropoiesis, due to a defect in the maturation of red blood cells, and die by embryonic day 16 (E16). [5][6][7] Several other members of the KLF family, including KLF2 (lung Krüppel-like factor, LKLF), also are expressed in erythroid cells. [8][9][10][11] Based on phylogenetic analyses, the zinc finger domains of KLF2 and EKLF are very similar. 1,2,12,13 KLF2 Ϫ/Ϫ mice die between E12.5 and E14.5 due to heart failure and severe hemorrhaging, caused by defects in vascular endothelial cells and in stabilization of immature vessels by recruited smooth muscle cells. 14,15 Prior to E12.5, KLF2 Ϫ/Ϫ embryos have normal vasculogenesis and angiogenesis. 14,15 KLF2 also plays an important role in hematopoietic cell biology. We reported that KLF2 is essential for primitive (embryonic yolk sac) erythropoiesis and positively regulates the embryonic ␤-like globin genes in vivo. E10.5 KLF2 Ϫ/Ϫ primitive erythroid cells have abnormal morphology. 9 KLF2 also regulates T-cell activation. Deficiency of KLF2 leads to a decrease in the peripheral T-cell pool 16 due to defective thymocyte emigration. 17 Overexpression of KLF2 in mice inhibits proinflammatory activation of peripheral blood monocytes. 18 It was initially reported that EKLF does not affect embryonic/ fetal globin gene expression. Interestingly, however, EKLF is expressed very early in mouse and chicken development, as early as the primitive streak stage, fo...

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.

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