BACKGROUND
vasculogenesis is a dynamic process in mammalian embryonic development.
endothelial precursors, known as angioblasts, initiate intraembryonic vascular development by murine embryonic day  <dig>   forming a primary capillary plexus   <cit> .
during maturation, endothelial cells recruit mesenchymal cells or vascular smooth muscle cell  progenitors to the surface of the vessels, and these cells organize into layers around the tube to support it   <cit> .
by e <dig> , endothelial cells also promote and maintain vsmc differentiation  <cit> .
krüppel-like factor  <dig>  is a zinc finger dna binding protein with essential roles in vascular endothelial biology.
the gene is expressed in endothelial cells in the developing mouse as early as e <dig>   <cit> .
klf <dig> knockout mice die in utero between e <dig>  and e <dig> , and the time of death is dependent on the genetic background  <cit> .
angiogenesis and vasculogenesis appear grossly normal in viable e <dig>  klf2-/- mice.
kuo et al.
generated klf <dig> knockout embryos, and concluded that death is due to hemorrhaging and a lack of integrity in the smooth muscle layers that surround vessels from around e <dig>   <cit> .
our previous studies show that in the absence of klf <dig>  the dorsal aortae are abnormal in e <dig>  fvb/n mice.
the endothelial cell layer lacks integrity and there are erythroid cells outside of the aortae  <cit> .
in another study, wu et al.
showed that klf2-/- embryos have normal endothelial cell development, but a failure of mural cells to migrate around endothelial cells to stabilize blood vessels  <cit> .
recent findings suggest that klf <dig> plays an important role in endothelial barrier function in adult mice.
it positively regulates expression of the tight junction protein occludin and modification of myosin light chain that is important for the integrity of the endothelial layer and to avoid vascular leakage  <cit> .
krüppel-like factor  <dig>  is a member of the krüppel-like transcription factor family, and is ~90% similar to klf <dig> in its zinc finger dna binding domain, suggesting the factors could have common target sequences.
klf <dig> is expressed in mesenchymal tissue, endothelium and epithelium by e <dig>   <cit> , and is essential for skin barrier function during development  <cit> .
klf <dig> knockout mice die soon after birth  <cit> .
in tamoxifen-inducible klf4-/- adult mice, vascular injury-induced repression of smooth muscle cell markers is delayed, thus indicating that klf <dig> controls phenotypic switching of vascular smooth muscle cells  <cit> .
vascular abnormalities have not been reported during the early embryonic development of klf4-/- mice.
in tissue culture, klf <dig> plays a role as a molecular transducer of fluid shear forces, thus directly or indirectly regulating a number of endothelial genes including endothelial nitric oxide synthase  and endothelin  <cit> .
klf <dig> is induced by laminar shear stress in human umbilical vein endothelial cells  and transactivates the enos and thrombomodulin  promoters.
tm and enos are important in vascular tone regulation and maintenance of intact endothelium  <cit> .
klf <dig> and klf <dig> are induced by shear stress and activated by the mek5/mef <dig> signaling pathway.
using genome wide transcriptional profiling of huvec cells overexpressing klf <dig>  klf <dig> or constitutively active mek <dig>  it was shown that ~60% of the genes activated by mek <dig> were also regulated by either klf <dig> or klf <dig>  these studies suggest that there is mechanistic and functional conservation between klf <dig> and klf <dig> in vascular endothelial cells  <cit> .
the combined roles of klf <dig> and klf <dig> have thus far been studied only in in vitro endothelial models.
because the two factors are evolutionarily closely related, it was of interest to determine if they had overlapping roles in embryonic development.
this study shows for the first time that there are interactions between the klf <dig> and klf <dig> genes during vascular development.
RESULTS
klf2-/-klf4-/- embryos show hemorrhaging in the cranial region
klf2-/- embryos die between e <dig>  and e <dig>   <cit>  and klf4-/- mice die perinatally  <cit> .
of the  <dig> embryos obtained from nine klf2+/-klf4+/- matings, at least  <dig> e <dig>  klf2-/-klf4-/- embryos were expected to be obtained.
however, no live klf2-/-klf4-/- embryos were observed at e <dig> , and the necrotic klf2-/-klf4-/- embryos that were recovered lacked integrity, and could not successfully be processed for sectioning.
nevertheless, in these e <dig>  klf2-/-klf4-/- embryos, gross hemorrhaging was evident in the head and trunk , suggesting that this might be a contributing factor in their early death.
surprisingly, of the  <dig> klf2-/-klf4+/- embryos obtained, only one embryo had a beating heart and the other  <dig> were not viable.
gross hemorrhaging was also observed in a majority of these embryos.
at e <dig> , klf2-/- embryos were viable as evidenced by a beating heart, and did not exhibit gross cranial hemorrhaging.
this thus suggests that the additional loss of one klf <dig> allele from klf2-/- embryos results in the hemorrhaging phenotype, implying an interaction between the klf <dig> and klf <dig> genes.
to further investigate the vascular phenotype of klf2-/-klf4-/- compared to klf <dig> and klf <dig> single knockout embryos, macroscopic examination of intact embryos and light microscopic examination of tissue sections was performed to assess morphological abnormalities.
e <dig>  embryos were collected from klf2+/-klf4+/- matings.
the expected numbers of all genotypes were obtained, yielding wt , klf2+/-klf4+/- , klf2-/- , klf4-/- , klf2-/-klf4+/- , klf2+/-klf4-/- , and klf2-/-klf4-/-  embryos.
all of the e <dig>  klf2-/-klf4-/- embryos were viable, as evidenced by a heartbeat.
cranial hemorrhaging was not observed in the wt , klf2-/-  or klf4-/-  embryos.
klf2-/-klf4+/- and klf2+/-klf4-/- embryos also did not show gross hemorrhaging .
one of the klf2-/-klf4-/- embryos showed gross hemorrhaging macroscopically, in the cranial region, beginning rostrally at the level of optic vesicle and ending caudally at the level of the first branchial arch .
gross and microscopic examination confirmed that hemorrhaging in e <dig>  klf2-/-klf4-/- embryos is confined to the primary head vein  and the rostral portion of the anterior cardinal vein, into which the phv drains.
bleeding penetrates at some levels, all the way to the midline infiltrating the supporting cranial mesenchyme, a precursor tissue to vascular smooth muscle.
gross hemorrhaging in klf2-/-klf4-/- embryos is more severe and occurs in vessels other than the phv by e <dig> , consistent with the time of death.
tissue-sections of the phv at the level of the optic vesicle reveal a continuous endothelium in e <dig>  wt  and klf2-/-  embryos.
in klf2-/-klf4-/- embryos, there are apparent gaps between adjoining endothelial cells .
endothelial disruption of the phv, which was not evident at the gross level, is also seen at the microscopic level in two of the four klf2-/-klf4+/- embryos that were examined .
this phenotype is variable, and the other two e <dig>  klf2-/-klf4+/- embryos appeared like wild-type, having no phv phenotype.
the presence of apparent gaps in the endothelial layer suggests a lack of vascular integrity.
no abnormal phenotype was seen in klf2+/-klf4-/- embryos , suggesting that complete klf <dig> ablation is required for this abnormal vascular phenotype.
the fact that the klf2-/-klf4-/- and klf2-/-klf4+/- phenotypes are more severe than klf2-/- indicates that klf <dig> plays a larger role in vascular development than previously recognized and is complemented by klf <dig>  serial tissue sections of entire e <dig>  embryos were examined for this study, and the abnormal vascular phenotype in klf2-/-klf4-/- and some klf2-/-klf4+/- embryos is observed only in the primary head vein.
the primary head vein is one of the major blood vessels at this stage of development.
although the phv is the only vessel to exhibit hemorrhaging at e <dig> , based on the gross hemorrhaging observed by e <dig> , other vessels in klf2-/-klf4-/- embryos lack integrity by this later time point.
electron micrographs confirm disruption of the endothelial layer of the primary head vein in klf2-/-klf4-/- embryos at e <dig>
the apparent endothelial disruption at the primary head vein observed in e <dig>  klf2-/-klf4-/- embryos was more completely characterized using transmission electron microscopy .
electron micrographs of the phv at the level of the optic vesicle are shown in figure  <dig>  in the wt, the endothelium is continuous with gaps ≤ 5 μm .
the typical flattened spindle shape of the endothelial cells seen at the light level is confirmed.
the klf4-/- embryo also has endothelium that is generally continuous with gaps that are indistinguishable from wt .
klf2-/- embryos have a modest and variable phenotype.
one klf2-/- phv has no visible abnormalities of the endothelium, with gaps appearing identical to the wt and klf4-/- embryos .
another klf2-/- shows multiple ≤ 8 μm gaps along the medial aspect of the vessel .
endothelial cells are present and outline the lumen of the phv.
klf2-/-klf4-/- embryos have endothelial disruption and more frequent gaps ≤ 20 μm on both the medial and lateral aspects of the fragmented vessel wall .
furthermore, the endothelial cells in the klf2-/-klf4-/- vessels are more bulbous in shape compared to klf2-/-, klf4-/- and wt.
these findings confirm the disruption of the endothelial layer of the phv observed at the light microscopy level in e <dig>  klf2-/-klf4-/- embryos.
in all four genotypes tested, mesenchymal cells are found near the phv at e <dig> , but muscle cells have not yet formed a continuous layer surrounding the vessel .
klf <dig> and klf <dig> regulate enos mrna expression at e <dig>
to begin to elucidate the molecular mechanism by which klf <dig> and klf <dig> ablation results in endothelial disruption, the amounts of mrna for endothelial cell regulators were quantified.
genes were selected for their established role in vascular development, a similar ko phenotype to klf2-/-klf4-/-, the presence of potential klf <dig> and klf <dig> binding sites  in the proximal promoter  <cit> , and a high ratio of expression in endothelial compared to other cell types at e <dig> .
expression of enos mrna in e <dig>  mouse embryos was quantified and normalized to the ubiquitously-expressed housekeeping gene, cyclophilin a .
while no difference in enos mrna expression is noted between wt and klf2-/-, enos mrna expression in klf2-/-klf4-/- is significantly lower than in wt , klf2+/-klf4+/-  and klf2-/-  embryos using a two-tailed student t-test.
additionally, using the same statistical test, enos expression in klf2-/-klf4+/- is significantly lower than in wt , klf2+/-klf4+/-  and klf2-/- embryos  .
unfortunately, klf4-/- embryos were not obtained for the gene expression studies; we cannot rule out the possibility that klf <dig> knockout alone affects endothelial cell gene regulation.
the data indicate that both klf <dig> and klf <dig> are required for enos regulation.
interestingly, lower enos mrna expression correlates with the more severe phenotype in klf2-/-klf4+/- and klf2-/-klf4-/- embryos compared to the other genotypes.
klf <dig> and klf <dig> positively affect the expression of enos, which is a regulator of endothelial homeostasis and vasculogenesis.
enos-/- mice are not embryonic lethal and their phenotype is indistinguishable from enos+/- and wild-type mice in general appearance and histology  <cit> .
thus, other genes downstream of klf <dig> and klf <dig> must also contribute to the embryonic lethality of klf2-/-klf4-/-.
vegfr <dig>  and occludin  mrna expression is significantly reduced in klf2-/-klf4-/- and klf2-/-klf4+/- embryos compared to wt.
the decrease in these mrnas is modest , however vegfr <dig> and occludin are not endothelial cell-specific, and their reduction in endothelial cells may be more pronounced.
vegfr <dig> induces enos expression  <cit>  and occludin is a tight junction protein  <cit> .
therefore, the reduced expression of these genes could logically contribute to the observed vascular integrity phenotype.
the other genes tested were endothelin- <dig>  thrombomodulin- <dig> and pecam <dig>  none of these genes showed significant differences in mrna expression in the klf2-/-klf4-/- or klf2-/-klf4+/- compared to wt or klf2-/- embryos.
therefore, it is not likely that these genes are responsible for the diminished vascular integrity observed in klf2-/-klf4-/- and klf2-/-klf4+/- embryos.
discussion
at e <dig> , klf2-/-klf4-/- embryos exhibit variable macroscopic and uniform microscopic hemorrhaging, and these are sometimes observed in klf2-/-klf4+/- embryos.
klf2-/-klf4-/- and most klf2-/-klf4+/- embryos die by e <dig> , which is earlier than either single knockout.
similarly, klf1-/-klf2-/- embryos also die sooner than either single knockout  <cit> .
the kfl2-/-klf4+/- phenotype is more variable and less severe than klf2-/-klf4-/-, indicating that the loss of an additional klf <dig> allele negatively impacts mouse embryonic vascular development.
interestingly, klf2+/-klf4-/- embryos have a less severe phenotype than either klf2-/-klf4+/- or klf2-/-klf4-/- embryos, indicating that hemorrhaging at e <dig>  occurs only with the complete loss of klf <dig>  a similar gene dosage effect is also evident in double mutant hoxa- <dig> and hoxd- <dig> mice  <cit> .
hoxa-13-/-hoxd-13-/- and hoxa-13-/-hoxd-13+/- mutations are embryonic lethal.
hoxa-13+/-hoxd-13-/- mice reach adulthood but have more severe abnormalities of the genitourinary and digestive systems than seen in hoxa-13-/- or hoxa-13+/-hoxd-13+/- double heterozygote mice.
interestingly, loss of blood vessel integrity is observed only in the primary head vein in e <dig>  klf2-/-klf4-/- embryos.
the significance of this observation is somewhat diminished by the fact that many vessels throughout the klf2-/-klf4-/- embryo exhibit gross hemorrhaging by e <dig> , coincident with embryonic demise.
the primary head vein is one of the major blood vessels at e <dig> , but there is no evidence that it is subject to higher fluid shear force.
the most prominent effect on gene expression that we observed in klf2-/-klf4-/- compared to klf2-/- and wt e <dig>  embryos is a reduction in enos mrna.
enos regulates many processes relevant to vasculogenesis, including vasodilation, vascular smooth muscle tone, and endothelial homeostasis  <cit> .
the roles of enos and nitric oxide in embryonic vascular development are less well-studied than in the adult.
however, recently a role for vegf-mediated regulation of enos in angioblast and embryonic endothelial cell proliferation has been discovered  <cit> .
in e <dig>  enos-gfp embryos, gfp can be detected in the vessels supplying the head, suggesting that enos could be expressed in primary head vein endothelial cells  <cit> .
blood vessels in embryos lacking klf <dig> and klf <dig> might not be able to withstand increasing fluid shear stress leading to their disruption, and this could be due in part to a reduction in enos.
the lack of vascular integrity observed in klf2-/-klf4-/- embryos in this study is apparently not due simply to a lack of vascular smooth muscle or mural cell recruitment to vessels, because there is not yet a continuous layer of mesenchymal cells surrounding the primary head vein at e <dig> .
although enos can be dispensable in early embryos, some enos-/- embryos die between e <dig>  and e <dig> , coincident with the time of death of klf2-/-klf4-/- embryos  <cit> .
in the absence of klf <dig> and klf <dig> it is likely that a host of other endothelial regulators are down-regulated, in addition to enos, exacerbating the effect.
occludin and vegfr <dig> mrna are also reduced in e <dig>  embryos lacking klf <dig> and klf <dig>  occludin is a tight junction protein, and its role in vascular integrity has been established  <cit> .
complete ablation of vegfr <dig> leads to embryonic lethality and loss of vessel development  <cit> .
down-regulation of vegfr <dig> in klf2-/-klf4-/- mice may further diminish enos expression, because vegf signaling induces enos  <cit> .
the data suggest that the loss of both klf <dig> and klf <dig> creates a situation where enos deficiency cannot be compensated, due to disruption of other endothelial regulators, resulting in loss of vascular integrity.
in cultured huvec cells, overexpression of klf <dig> positively regulates the enos gene  <cit> .
in our study in e <dig>  mouse embryos, enos expression is regulated coordinately by klf <dig> and klf <dig>  but is not diminished in the absence of klf <dig> alone.
lee et al.
also did not observe a reduction in enos mrna in klf <dig> endothelial conditional knockout mice at e <dig>   <cit> .
similarly, when the klf <dig> gene is conditionally deleted in adult mice, occludin expression is reduced  <cit> , but occludin mrna is reduced in klf2-/-klf4-/- but not in klf2-/- embryos, compared to wt.
evidently, klf <dig> can partially compensate for klf <dig> in regulating the enos and occludin genes in embryonic vascular endothelial cells, but not in the adult mouse or in tissue culture systems.
a limitation of this work is the use of knockout mice lacking expression of klf <dig> and klf <dig> in all cell types, rather than just endothelial cells.
we cannot rule out the possibility that a lack of expression of klf <dig> or klf <dig> in cells other than endothelial cells may contribute to the vascular phenotype.
for example, in adult mice, klf <dig> and klf <dig> are expressed in monocytes and macrophages  <cit> ; it is not known if they are expressed in primitive phagocytes, and whether this could have a role in vasculogenesis.
furthermore, klf <dig> is expressed in vascular smooth muscle cells  <cit> , and could potentially be expressed and have a function in mesenchymal precursors to these cells at e <dig> .
however, only endothelial cells are specifically known to express both klf <dig> and klf <dig> at e <dig> , so the most parsimonious explanation is that these genes interact in endothelial cells, resulting in the phenotype exhibited in double mutant embryos.
in the future, a double conditional knockout mouse model could be used to determine if klf <dig> and klf <dig> have cell autonomous roles in the vascular endothelium.
CONCLUSIONS
this study begins to define the roles of klf <dig> and klf <dig> in the embryonic development of blood vessels.
the two genes interact to maintain an intact endothelial layer.
klf <dig> and klf <dig> positively regulate the enos, vegfr <dig> and occludin genes, and this may be required to establish or maintain vascular integrity.
klf <dig> and klf <dig> could regulate these genes through direct or indirect mechanisms, and this is a question for future study.

 vasculogenesis is a dynamic process in mammalian embryonic development. endothelial precursors, known as angioblasts, initiate intraembryonic vascular development by murine embryonic day forming a primary capillary plexus. during maturation, endothelial cells recruit mesenchymal cells or vascular smooth muscle cell progenitors to the surface of the vessels, and these cells organize into layers around the tube to support it. by e, endothelial cells also promote and maintain vsmc differentiation. krüppel-like factor is a zinc finger dna binding protein with essential roles in vascular endothelial biology. the gene is expressed in endothelial cells in the developing mouse as early as e. klf knockout mice die in utero between e and e, and the time of death is dependent on the genetic background. angiogenesis and vasculogenesis appear grossly normal in viable e klf2-/- mice. kuo et al. generated klf knockout embryos, and concluded that death is due to hemorrhaging and a lack of integrity in the smooth muscle layers that surround vessels from around e. our previous studies show that in the absence of klf the dorsal aortae are abnormal in e fvb/n mice. the endothelial cell layer lacks integrity and there are erythroid cells outside of the aortae. in another study, wu et al. showed that klf2-/- embryos have normal endothelial cell development, but a failure of mural cells to migrate around endothelial cells to stabilize blood vessels. recent findings suggest that klf plays an important role in endothelial barrier function in adult mice. it positively regulates expression of the tight junction protein occludin and modification of myosin light chain that is important for the integrity of the endothelial layer and to avoid vascular leakage. krüppel-like factor is a member of the krüppel-like transcription factor family, and is ~90% similar to klf in its zinc finger dna binding domain, suggesting the factors could have common target sequences. klf is expressed in mesenchymal tissue, endothelium and epithelium by e, and is essential for skin barrier function during development. klf knockout mice die soon after birth. in tamoxifen-inducible klf4-/- adult mice, vascular injury-induced repression of smooth muscle cell markers is delayed, thus indicating that klf controls phenotypic switching of vascular smooth muscle cells. vascular abnormalities have not been reported during the early embryonic development of klf4-/- mice. in tissue culture, klf plays a role as a molecular transducer of fluid shear forces, thus directly or indirectly regulating a number of endothelial genes including endothelial nitric oxide synthase and endothelin. klf is induced by laminar shear stress in human umbilical vein endothelial cells and transactivates the enos and thrombomodulin promoters. tm and enos are important in vascular tone regulation and maintenance of intact endothelium. klf and klf are induced by shear stress and activated by the mek5/mef signaling pathway. using genome wide transcriptional profiling of huvec cells overexpressing klf klf or constitutively active mek it was shown that ~60% of the genes activated by mek were also regulated by either klf or klf these studies suggest that there is mechanistic and functional conservation between klf and klf in vascular endothelial cells. the combined roles of klf and klf have thus far been studied only in in vitro endothelial models. because the two factors are evolutionarily closely related, it was of interest to determine if they had overlapping roles in embryonic development. this study shows for the first time that there are interactions between the klf and klf genes during vascular development.  klf2-/-klf4-/- embryos show hemorrhaging in the cranial region klf2-/- embryos die between e and e and klf4-/- mice die perinatally. of the embryos obtained from nine klf2+/-klf4+/- matings, at least e klf2-/-klf4-/- embryos were expected to be obtained. however, no live klf2-/-klf4-/- embryos were observed at e, and the necrotic klf2-/-klf4-/- embryos that were recovered lacked integrity, and could not successfully be processed for sectioning. nevertheless, in these e klf2-/-klf4-/- embryos, gross hemorrhaging was evident in the head and trunk, suggesting that this might be a contributing factor in their early death. surprisingly, of the klf2-/-klf4+/- embryos obtained, only one embryo had a beating heart and the other were not viable. gross hemorrhaging was also observed in a majority of these embryos. at e, klf2-/- embryos were viable as evidenced by a beating heart, and did not exhibit gross cranial hemorrhaging. this thus suggests that the additional loss of one klf allele from klf2-/- embryos results in the hemorrhaging phenotype, implying an interaction between the klf and klf genes. to further investigate the vascular phenotype of klf2-/-klf4-/- compared to klf and klf single knockout embryos, macroscopic examination of intact embryos and light microscopic examination of tissue sections was performed to assess morphological abnormalities. e embryos were collected from klf2+/-klf4+/- matings. the expected numbers of all genotypes were obtained, yielding wt, klf2+/-klf4+/-, klf2-/-, klf4-/-, klf2-/-klf4+/-, klf2+/-klf4-/-, and klf2-/-klf4-/- embryos. all of the e klf2-/-klf4-/- embryos were viable, as evidenced by a heartbeat. cranial hemorrhaging was not observed in the wt, klf2-/- or klf4-/- embryos. klf2-/-klf4+/- and klf2+/-klf4-/- embryos also did not show gross hemorrhaging. one of the klf2-/-klf4-/- embryos showed gross hemorrhaging macroscopically, in the cranial region, beginning rostrally at the level of optic vesicle and ending caudally at the level of the first branchial arch. gross and microscopic examination confirmed that hemorrhaging in e klf2-/-klf4-/- embryos is confined to the primary head vein and the rostral portion of the anterior cardinal vein, into which the phv drains. bleeding penetrates at some levels, all the way to the midline infiltrating the supporting cranial mesenchyme, a precursor tissue to vascular smooth muscle. gross hemorrhaging in klf2-/-klf4-/- embryos is more severe and occurs in vessels other than the phv by e, consistent with the time of death. tissue-sections of the phv at the level of the optic vesicle reveal a continuous endothelium in e wt and klf2-/- embryos. in klf2-/-klf4-/- embryos, there are apparent gaps between adjoining endothelial cells. endothelial disruption of the phv, which was not evident at the gross level, is also seen at the microscopic level in two of the four klf2-/-klf4+/- embryos that were examined. this phenotype is variable, and the other two e klf2-/-klf4+/- embryos appeared like wild-type, having no phv phenotype. the presence of apparent gaps in the endothelial layer suggests a lack of vascular integrity. no abnormal phenotype was seen in klf2+/-klf4-/- embryos, suggesting that complete klf ablation is required for this abnormal vascular phenotype. the fact that the klf2-/-klf4-/- and klf2-/-klf4+/- phenotypes are more severe than klf2-/- indicates that klf plays a larger role in vascular development than previously recognized and is complemented by klf serial tissue sections of entire e embryos were examined for this study, and the abnormal vascular phenotype in klf2-/-klf4-/- and some klf2-/-klf4+/- embryos is observed only in the primary head vein. the primary head vein is one of the major blood vessels at this stage of development. although the phv is the only vessel to exhibit hemorrhaging at e, based on the gross hemorrhaging observed by e, other vessels in klf2-/-klf4-/- embryos lack integrity by this later time point. electron micrographs confirm disruption of the endothelial layer of the primary head vein in klf2-/-klf4-/- embryos at e the apparent endothelial disruption at the primary head vein observed in e klf2-/-klf4-/- embryos was more completely characterized using transmission electron microscopy. electron micrographs of the phv at the level of the optic vesicle are shown in figure  in the wt, the endothelium is continuous with gaps ≤ 5 μm. the typical flattened spindle shape of the endothelial cells seen at the light level is confirmed. the klf4-/- embryo also has endothelium that is generally continuous with gaps that are indistinguishable from wt. klf2-/- embryos have a modest and variable phenotype. one klf2-/- phv has no visible abnormalities of the endothelium, with gaps appearing identical to the wt and klf4-/- embryos. another klf2-/- shows multiple ≤ 8 μm gaps along the medial aspect of the vessel. endothelial cells are present and outline the lumen of the phv. klf2-/-klf4-/- embryos have endothelial disruption and more frequent gaps ≤ 20 μm on both the medial and lateral aspects of the fragmented vessel wall. furthermore, the endothelial cells in the klf2-/-klf4-/- vessels are more bulbous in shape compared to klf2-/-, klf4-/- and wt. these findings confirm the disruption of the endothelial layer of the phv observed at the light microscopy level in e klf2-/-klf4-/- embryos. in all four genotypes tested, mesenchymal cells are found near the phv at e, but muscle cells have not yet formed a continuous layer surrounding the vessel. klf and klf regulate enos mrna expression at e to begin to elucidate the molecular mechanism by which klf and klf ablation results in endothelial disruption, the amounts of mrna for endothelial cell regulators were quantified. genes were selected for their established role in vascular development, a similar ko phenotype to klf2-/-klf4-/-, the presence of potential klf and klf binding sites in the proximal promoter, and a high ratio of expression in endothelial compared to other cell types at e. expression of enos mrna in e mouse embryos was quantified and normalized to the ubiquitously-expressed housekeeping gene, cyclophilin a. while no difference in enos mrna expression is noted between wt and klf2-/-, enos mrna expression in klf2-/-klf4-/- is significantly lower than in wt, klf2+/-klf4+/- and klf2-/- embryos using a two-tailed student t-test. additionally, using the same statistical test, enos expression in klf2-/-klf4+/- is significantly lower than in wt, klf2+/-klf4+/- and klf2-/- embryos. unfortunately, klf4-/- embryos were not obtained for the gene expression studies; we cannot rule out the possibility that klf knockout alone affects endothelial cell gene regulation. the data indicate that both klf and klf are required for enos regulation. interestingly, lower enos mrna expression correlates with the more severe phenotype in klf2-/-klf4+/- and klf2-/-klf4-/- embryos compared to the other genotypes. klf and klf positively affect the expression of enos, which is a regulator of endothelial homeostasis and vasculogenesis. enos-/- mice are not embryonic lethal and their phenotype is indistinguishable from enos+/- and wild-type mice in general appearance and histology. thus, other genes downstream of klf and klf must also contribute to the embryonic lethality of klf2-/-klf4-/-. vegfr and occludin mrna expression is significantly reduced in klf2-/-klf4-/- and klf2-/-klf4+/- embryos compared to wt. the decrease in these mrnas is modest, however vegfr and occludin are not endothelial cell-specific, and their reduction in endothelial cells may be more pronounced. vegfr induces enos expression and occludin is a tight junction protein. therefore, the reduced expression of these genes could logically contribute to the observed vascular integrity phenotype. the other genes tested were endothelin- thrombomodulin- and pecam none of these genes showed significant differences in mrna expression in the klf2-/-klf4-/- or klf2-/-klf4+/- compared to wt or klf2-/- embryos. therefore, it is not likely that these genes are responsible for the diminished vascular integrity observed in klf2-/-klf4-/- and klf2-/-klf4+/- embryos. discussion at e, klf2-/-klf4-/- embryos exhibit variable macroscopic and uniform microscopic hemorrhaging, and these are sometimes observed in klf2-/-klf4+/- embryos. klf2-/-klf4-/- and most klf2-/-klf4+/- embryos die by e, which is earlier than either single knockout. similarly, klf1-/-klf2-/- embryos also die sooner than either single knockout. the kfl2-/-klf4+/- phenotype is more variable and less severe than klf2-/-klf4-/-, indicating that the loss of an additional klf allele negatively impacts mouse embryonic vascular development. interestingly, klf2+/-klf4-/- embryos have a less severe phenotype than either klf2-/-klf4+/- or klf2-/-klf4-/- embryos, indicating that hemorrhaging at e occurs only with the complete loss of klf a similar gene dosage effect is also evident in double mutant hoxa- and hoxd- mice. hoxa-13-/-hoxd-13-/- and hoxa-13-/-hoxd-13+/- mutations are embryonic lethal. hoxa-13+/-hoxd-13-/- mice reach adulthood but have more severe abnormalities of the genitourinary and digestive systems than seen in hoxa-13-/- or hoxa-13+/-hoxd-13+/- double heterozygote mice. interestingly, loss of blood vessel integrity is observed only in the primary head vein in e klf2-/-klf4-/- embryos. the significance of this observation is somewhat diminished by the fact that many vessels throughout the klf2-/-klf4-/- embryo exhibit gross hemorrhaging by e, coincident with embryonic demise. the primary head vein is one of the major blood vessels at e, but there is no evidence that it is subject to higher fluid shear force. the most prominent effect on gene expression that we observed in klf2-/-klf4-/- compared to klf2-/- and wt e embryos is a reduction in enos mrna. enos regulates many processes relevant to vasculogenesis, including vasodilation, vascular smooth muscle tone, and endothelial homeostasis. the roles of enos and nitric oxide in embryonic vascular development are less well-studied than in the adult. however, recently a role for vegf-mediated regulation of enos in angioblast and embryonic endothelial cell proliferation has been discovered. in e enos-gfp embryos, gfp can be detected in the vessels supplying the head, suggesting that enos could be expressed in primary head vein endothelial cells. blood vessels in embryos lacking klf and klf might not be able to withstand increasing fluid shear stress leading to their disruption, and this could be due in part to a reduction in enos. the lack of vascular integrity observed in klf2-/-klf4-/- embryos in this study is apparently not due simply to a lack of vascular smooth muscle or mural cell recruitment to vessels, because there is not yet a continuous layer of mesenchymal cells surrounding the primary head vein at e. although enos can be dispensable in early embryos, some enos-/- embryos die between e and e, coincident with the time of death of klf2-/-klf4-/- embryos. in the absence of klf and klf it is likely that a host of other endothelial regulators are down-regulated, in addition to enos, exacerbating the effect. occludin and vegfr mrna are also reduced in e embryos lacking klf and klf occludin is a tight junction protein, and its role in vascular integrity has been established. complete ablation of vegfr leads to embryonic lethality and loss of vessel development. down-regulation of vegfr in klf2-/-klf4-/- mice may further diminish enos expression, because vegf signaling induces enos. the data suggest that the loss of both klf and klf creates a situation where enos deficiency cannot be compensated, due to disruption of other endothelial regulators, resulting in loss of vascular integrity. in cultured huvec cells, overexpression of klf positively regulates the enos gene. in our study in e mouse embryos, enos expression is regulated coordinately by klf and klf but is not diminished in the absence of klf alone. lee et al. also did not observe a reduction in enos mrna in klf endothelial conditional knockout mice at e. similarly, when the klf gene is conditionally deleted in adult mice, occludin expression is reduced, but occludin mrna is reduced in klf2-/-klf4-/- but not in klf2-/- embryos, compared to wt. evidently, klf can partially compensate for klf in regulating the enos and occludin genes in embryonic vascular endothelial cells, but not in the adult mouse or in tissue culture systems. a limitation of this work is the use of knockout mice lacking expression of klf and klf in all cell types, rather than just endothelial cells. we cannot rule out the possibility that a lack of expression of klf or klf in cells other than endothelial cells may contribute to the vascular phenotype. for example, in adult mice, klf and klf are expressed in monocytes and macrophages; it is not known if they are expressed in primitive phagocytes, and whether this could have a role in vasculogenesis. furthermore, klf is expressed in vascular smooth muscle cells, and could potentially be expressed and have a function in mesenchymal precursors to these cells at e. however, only endothelial cells are specifically known to express both klf and klf at e, so the most parsimonious explanation is that these genes interact in endothelial cells, resulting in the phenotype exhibited in double mutant embryos. in the future, a double conditional knockout mouse model could be used to determine if klf and klf have cell autonomous roles in the vascular endothelium.  this study begins to define the roles of klf and klf in the embryonic development of blood vessels. the two genes interact to maintain an intact endothelial layer. klf and klf positively regulate the enos, vegfr and occludin genes, and this may be required to establish or maintain vascular integrity. klf and klf could regulate these genes through direct or indirect mechanisms, and this is a question for future study. 
krüppel-like factor plays an important role in vessel maturation during embryonic development. electron micrographs confirm that there are gaps in the phv endothelial layer in e klf2-/-klf4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to klf2-/- and wild-type. endothelial breaks are observed in a major vessel, the primary head vein, in klf2-/-klf4-/- embryos at e. klf and klf positively regulate the enos, vegfr and occludin genes. this study begins to define the roles of klf and klf in the embryonic development of blood vessels. klf2-/-klf4-/- embryos die by e, which is earlier than either single knockout. down-regulation of these genes in klf2-/-klf4-/- embryos may result in the observed loss of vascular integrity. in adult mice, klf regulates expression of the tight junction protein occludin, which may allow klf to maintain vascular integrity. it indicates that the two genes interact to maintain an intact endothelial layer. vegfr an enos inducer, and occludin, a tight junction protein, gene expression are also reduced in e klf2-/-klf4-/- compared to klf2-/- and wt embryos. klf2klf4gene interactionsembryonic vascular integrityendothelial cell development
 krüppel-like factor plays an important role in vessel maturation during embryonic development. in adult mice, klf regulates expression of the tight junction protein occludin, which may allow klf to maintain vascular integrity. adult tamoxifen-inducible krüppel-like factor knockout mice have thickened arterial intima following vascular injury. the role of klf and the possible overlapping functions of klf and klf in the developing vasculature are not well-studied.  endothelial breaks are observed in a major vessel, the primary head vein, in klf2-/-klf4-/- embryos at e. klf2-/-klf4-/- embryos die by e, which is earlier than either single knockout. gross hemorrhaging of multiple vessels may be the cause of death. e 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 e. electron micrographs confirm that there are gaps in the phv endothelial layer in e klf2-/-klf4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to klf2-/- and wild-type. the amount of endothelial nitric oxide synthase mrna, which encodes an endothelial regulator, is reduced by 10-fold in e klf2-/-klf4-/- compared to klf2-/- and wt embryos. vegfr an enos inducer, and occludin, a tight junction protein, gene expression are also reduced in e klf2-/-klf4-/- compared to klf2-/- and wt embryos.  this study begins to define the roles of klf and klf in the embryonic development of blood vessels. it indicates that the two genes interact to maintain an intact endothelial layer. klf and klf positively regulate the enos, vegfr and occludin genes. down-regulation of these genes in klf2-/-klf4-/- embryos may result in the observed loss of vascular integrity. klf2klf4gene interactionsembryonic vascular integrityendothelial cell development 