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Keywords:

  • blood vessel;
  • cadherin;
  • cardiovascular system;
  • hematopoietic progenitors;
  • vasculature

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The ability to target gene deletion to a specific cellular compartment via the Cre/loxP system has been a powerful tool in the analysis of broadly expressed genes. Here, we report the generation of a transgenic mouse line in which expression of Cre-recombinase is under the regulatory control of the VE-Cadherin promoter. Temporal distribution and activity of the enzyme was evaluated with two independent Cre reporter lines. Histological analysis was performed throughout development and in the adult. Recombination of lox P sites with subsequent expression of β-galactosidase or GFP was detected as early as E7.5 in endothelial cells of the yolk sac. Progressive staining of the embryonic vasculature was noted from E8.5–13.5; however, more contiguous reporter expression was only seen by E14.5 onward in all endothelial compartments including arteries, veins, and capillaries. In addition, we found Cre activity in lymphatic endothelial cells. Unlike other endothelial-specific Cre mice, this model showed expression in the adult quiescent vasculature. Furthermore, the constitutive nature of the VE-Cadherin promoter in the adult can be advantageous for analysis of gene deletion in pathological settings. Developmental Dynamics 235:759–767, 2006. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The mouse is an excellent model organism in which to investigate the consequences of genetic manipulation within the vascular system. Conventional transgenic approaches have been extremely useful in exploring the contribution of specific genes to vascular morphogenesis, physiology, and pathology. These technologies have advanced our understanding of vascular biology allowing mechanistic insight into disease etiology and exploration of therapeutic strategies. More recently, the Cre/lox system has increased specificity and added flexibility to the manipulation of the mouse genome. The technology relies on use of the bacteriophage P1 Cre-recombinase that recognizes loxP sites (lox=locus of recombination) and removes intervening DNA sequences. Dependent on the expression pattern of the promoter driving Cre-recombinase, genetic deletion can be accomplished in somatic cells and their entire progeny. This property allows for lineage tracing and facilitates the cellular expansion of the mutation in the absence of active Cre expression (for a review see Nagy,2000).

Within the past 5 years, several transgenic lines have been developed to target Cre-recombinase expression in the vascular endothelium. These include: Tie-1 and Tie-2-Cre, PECAM-Cre, Flk-1-Cre, and, more recently, SCL-Cre (Terry et al.,1997; Licht et al.,2004; Gustasfsson et al.,2001; Kisanuki et al.,2001; Gothert et al.,2005). While these lines have been extremely useful in gene deletion studies, limitations related to expression in hematopoietic lineages and other cell types have confounded the aim to answer specific questions related to the endothelium. Furthermore, the activity of these promoters in the adult was not, for the most part, expressed in quiescent endothelium; making the potential use of a constitutive, or inducible Cre mouse counterpart, less attractive. Our goal was to generate a constitutive Cre transgenic model with specific expression in the differentiated endothelium of embryos and adults. Thus, we sought to take advantage of the VE-Cadherin promoter as an alternative for the development of a new endothelial Cre-line. Elements of the VE-Cadherin promoter have been well characterized and previous studies have demonstrated its activity in adult/quiescent endothelium (Gory et al.,1999; Hisatsune et al.,2005; Prandini et al.,2005).

Vascular endothelial cadherin (VE-Cadherin), also known as CD-144 and cadherin-5, is a transmembrane protein involved in endothelial homotypic cell adhesion (Lampugnani et al.,1995). In functional analogy to other cadherins, VE-Cadherin has been localized to adherens junctions in endothelial cells (Dejana,1996). Absence of VE-Cadherin by homologous recombination in mice results in embryonic lethality by E9.5 due to vascular abnormalities (Carmeliet et al.,1999; Gory-Faure et al.,1999). These findings support the essential role of this protein in the morphogenesis of the vascular system. In addition, VE-Cadherin has been implicated in modulation of flow, VEGF signaling, and vascular permeability within a variety of organs in adult mice (Calera et al.,2004; Corada et al.,2001; May et al.,2005; Venkiteswaran et al.,2002). These data combined with extensive expression analysis are supportive of a constitutive activity for this gene in adult endothelium during physiological and pathological conditions (Lambeng et al.,2005). Thus, the limitations of currently available endothelial Cre-lines combined with data on VE-Cadherin expression provided us with a compelling reason to generate VE-Cadherin-Cre transgenic mice for purposes of future genetic deletion studies.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

To generate the VE-Cadherin-Cre transgenic mouse, we fused the Cre-recombinase coding region to a regulatory region of the VE-Cadherin promoter (Fig. 1a). The region comprised 2.5 kb of the VE-Cadherin promoter upstream of the ATG site that has been previously shown to direct endothelium specific expression when fused to the reporter CAT (Gory et al.,1999). A total of five founders was obtained after microinjection of mouse oocytes. Two of these lines showed transmission to the germline and broad Cre-recombinase expression when tested by reverse transcriptase-polymerase chain reaction (RT-PCR) in several organs, consistent with expression in the endothelial compartment. To examine the spatio-temporal specificity of Cre expression, we crossed these lines to the Rosa26R (R26R) reporter (Soriano,1999) and Rosa-EGFPR lines (Mao et al.,2001). Mice resulting from these crosses had expected sized litters, mendelian frequency, and showed no signs of abnormalities in either embryonic stages or adult. Identification of genotype was performed by genomic PCR for the Rosa locus and presence of Cre (Fig. 1b). The R26R locus enables β-galactosidase expression after Cre-mediated excision of the neo-cassette, with noted expression in all cells in adult mice (Soriano,1999). Consequently, detection of LacZ is indicative of Cre expression and activity in either the positive cell or its progenitor.

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Figure 1. Generation of the VE-Cadherin-Cre R26R mice and characterization of Cre recombination. a: Schematic diagram of the VE-Cadherin-Cre transgene. b: Genotyping of transgenic animals. PCR was performed on yolk sac or tail DNA to determine the genotype. A 550-bp band corresponds to the wildtype (WT) allele and the 300-bp band corresponds to the R26R allele (R26R). A band of 700 bp indicates the presence of the Cre transgene. Lanes: 1, 100-bp ladder; 2, 5, 6, heterozygous for Rosa26R; 3, 8, homozygous R26R; 4, 7, wildtype animals. Only animals from lanes 5, 6, and 8 harbored the Cre-recombinase transgene. c: Percentage of endothelium positive for β-galactosidase. Two litters aged E10.5 and E12.5 were sorted by (a') FSC and SSC (gate R1) and (b') viability by lack of 7-AAD uptake (gate R2). Average viability (gated on R1+R2) was 25%. Of this viable population, cells were compared by PE-CD41 and APC-PECAM; isotype control in c'. d': Endothelial cells, phenotype PECAM+/CD41−, were sorted on gate R3. From the sorted population, cells were stained and counted for percentage positive of β-galactosidase staining (e'). Statistical analysis of average % positive across embryonic age E10.5 and E12.5, plus standard error, was completed and a Student's t-test (2-tailed heteroscedastic) was performed producing a P value of 0.4. Alternatively, endothelial cells of yolk sacs (f') and embryos (g') from E7.5 to adult were counted from histological sections for percentage positive of β-galactosidase staining (average number counted: 512 cells for age < E10.5, 1,314 cells for ≥ E10.5 across 2–4 embryos each age group).

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Efficiency of recombination in the endothelial compartment was assessed by FACS sorting of cells obtained from E10.5 and E12.5 embryos (Fig. 1c). As noted by β-gal activity, the percentage of β-gal positive cells increased over time. Cells were sorted for an endothelial phenotype, which we defined as PECAM+/CD41− (this procedure excludes blood cells that may co-express PECAM) (Baumann et al.,2004). CD45 (common leukocyte antigen) was not used as the majority of the blood cell population, at the chosen developmental timepoints, do not express CD45, but CD41 (Ferkowicz et al.,2003; Li et al.,2005; Mikkola et al.,2003). Evaluation of the LacZ reporter in the sorted PECAM+/CD41− population indicated that 64.9% (±8%) of embryonic endothelial cells showed Cre activity at E10.5; this number increased to 75.5% (±8%) by E12.5 (Fig. 1c, panel e'). In addition, morphometric counting of β-gal positive and negative endothelial cells within histological sections of yolk sac and embryos was also performed (Fig. 1c, panels f' and g'). Similar to the FACS findings, these data suggested progressive Cre activity during embryonic development with close to full penetrance (96.4%) by E14.5.

Whole-mount β-gal staining of mouse embryos revealed positive cells in the yolk sac by E7.5 (Fig. 2a). Intraembryonic labeling was noted by E8.5 in the heart primordium and dorsal aorta (Fig. 2b). By E9.5, staining had extended to small capillaries. However, not all endothelial cells were positive, providing an interrupted and rather spotty aspect to the vascular tree (Fig. 2c). Histological evaluation is consistent with this conclusion (Fig. 2d–f). In particular at E9.5, endothelial positivity is patchy in the dorsal aorta and positive cells are also found within the lumen (Fig. 2f). The percentage of reporter-positive endothelial cells increased throughout development (Fig. 2g–i). Unexpectedly, labeling of circulating cells was also found within yolk sac vessels by whole-mount staining and within the vascular lumen of sectioned embryos (Fig. 2j and l). β-gal staining of the embryonic vasculature was clearly penetrant by E14.5 both in the embryo and yolk sac (Fig. 2m–r). Because of recent reports indicating the expression of VE-Cadherin in the trophoblast population (Chang et al.,2005), we also evaluated the placenta for Cre activity. Our findings revealed Cre activity in the embryonic placental vessels of a positive embryo, but not in other cell types including the maternal vessels of the negative mother (Fig. 2o).

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Figure 2. Characterization of Cre activity during embryonic development in VE-Cadherin-Cre R26R embryos. a: A predominant population of cells within extraembryonic blood islands are LacZ positive in the E7.5 yolk sac (YS). b: Frontal view of E8.5 embryo. Arrow indicates the heart primordium (HP). Main aortic arch arteries are also evident. c: At E9.5, LacZ was detected in the aorta, intersomitic vessels, and developing capillaries. However, at this time expression is rather spotty. On the right is a Cre-negative littermate also stained for LacZ. d: Low-magnification frontal section of E9.5. e: Cross-section of E9.5 indicates vascular staining; arrows indicate β-gal-positive endothelium. f: High magnification of the dorsal aorta at E9.5 shows discontinuous endothelial staining (arrows) and arrowhead denotes negative endothelial cell. g: More contiguous staining was detected by E10.5. In this embryo, the aorta (A) and heart (H) are indicated. h: E12.5 lower limb (LL). i: Histological section of limb in h revealed LacZ-positive capillaries (arrow) among the condensing cartilage (cc) tissue of the developing digits. j: β-gal expression was evident throughout the primary plexus of the E8.5 YS. Arrows point to endothelium and arrowhead denotes circulating cells. k: The small and large vessels in the E12.5 yolk sac express β-gal. The placenta (P) is also positive. l: Histological section of E12.5 yolk sac. The arrow points to LacZ-positive endothelium and the arrowheads denote putative blood cells. m: E14.5 yolk sac. n: E16.5 yolk sac. o: Histological section of placenta demonstrates LacZ-positive embryonic vessels (E) versus maternal placental tissue (M). p: Histological section of E14.5 heart (H) reveals positive endothelial cells in the endocardium (arrow). q: Histological section of E14.5 lung demonstrating positive endothelium in lung vessels (arrows) r: Histological section of E14.5 nasal septum (C, cartilage) and surrounding tissue. Arrows point to β-gal-positive capillaries. s: EGFP-expressing vessels of adult abdominal muscle. t: EGFP-positive capillaries of the adult abdominal skeletal muscle (arrows). u: EGFP-positive limb vessels of E14.5.

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Investigation of the EGFP reporter line showed an identical pattern of expression (Fig. 2s–u). We did not find ectopic LacZ, nor expression of the reporter in Cre-negative mice, or variations in the pattern of Cre activity when the lines were crossed into C57Bl6J mice for 8 generations (data not shown).

Histologic evaluation of β-gal-stained mice at several embryonic stages revealed that Cre activity was equally penetrant in arteries, veins, and capillaries (Fig. 3a–f). Immunocolocalization with PECAM confirmed that, indeed, β-gal expression corresponded to endothelial cells (Fig. 3g–i). We also detected β-gal expression in the lymphatic vasculature. Specification of the lymphatic lineage is mediated by the transcription factor Prox-1 (Wigle et al.,2002). Onset of lymphangiogenesis in the mouse occurs at approximately E10 with the specification of a population of Prox-1-positive lymphatic endothelial progenitor cells that are visible as a polarized subpopulation in the endothelium of the cardinal veins. These cells ultimately bud and migrate from the veins to form the lymph sacs and, subsequently, the lymphatic vascular network. We found that Prox-1-positive cells in the dorsal wall of the cardinal vein did not express β-gal at E10.5 (Fig. 3j). However, lymphatic vessels identified by Prox-1 staining at E12.5 and subsequent stages showed consistent and contiguous β-gal expression, indicating Cre activity in lymphatic vessels (Fig. 3k–l). Thus, it appears that VE-Cadherin promoter activity is not present prior to the budding of lymphatic progenitor cells from the cardinal vein as assessed by β-gal expression.

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Figure 3. VE-Cadherin-Cre R26R mice express active Cre-recombinase in different branches of the vasculature. a: LacZ-positive artery (A) and vein (V) in the adult abdominal wall. b: Endothelium of E14.5 dorsal aorta (DA). c: Transversal section of medium-size artery in the adult kidney. d: Vein (V) in the E14.5 thymus is LacZ positive. e: Arrows point to LacZ-positive capillaries (c) of E14.5 ribs. f: LacZ-positive capillaries (c) of E14.5 choroid plexus. g–i: Colocalization with the endothelial maker PECAM. g: Dorsal aorta of E9.5. Arrowheads indicate PECAM location in brown. Note that at this time, β-gal positivity is restricted to the ventral side of the aorta. h: Low magnification of adult skeletal muscle. Arrows point to vessels that are positive for both β-gal and PECAM. i: High magnification of a vessel showing intercellular presence of PECAM (arrowheads) and LacZ positivity. j–l: β-gal staining in lymphatic endothelial cells. j: At E10.5, β-gal expression is detected in the anterior endothelium of the cardinal vein (CV) (Prox-1 negative). However Prox-1 positive cells (arrows indicating nuclear brown staining) are LacZ negative. k: By E12.5, β-gal expression is detected in Prox-1-positive lymphatic endothelial cells of the embryonic skin. Arrow denotes lymphatic vasculature (LV). l: β-gal expression remains in Prox-1-positive lymphatic endothelial cells at E15.5 (black arrows); expression is also visible in blood endothelium and in circulating cells (blue arrows).

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In adult mice, endothelium specific LacZ activity was fully penetrant in the heart including endocardial layers (data not shown), large and small vessels within the atria and ventricles (Fig. 4a–c). β-gal expression in the kidney glomeruli was noted following vibratome sectioning (Fig. 4d). LacZ was also contiguous in the endothelium of arteries and in the capillaries (Fig. 4e,f) of the kidney. The brain vasculature showed positive and specific staining in all vessels observed (Fig. 4g–i). Intense LacZ reaction was noted in the lung (Fig. 4j). Histological examination of vibratome sections revealed positive labeling in larger vessels and in the capillaries invested in the alveolar wall (Fig. 4k,l). No staining was noted in the smooth muscle or epithelia of bronchiolar units. In the intestine, LacZ activity was seen in the endothelium of vessels located in the mucosa, submucosa, and muscle layers (Fig. 4m–o). We also examined the uterus, mature retina, tongue, testis, mesentery, and pancreas. Evaluation of these tissues revealed uniform endothelial staining (Fig. 4p–u). β-gal expression in ovary, salivary gland, mammary gland, adrenal, skeletal muscle, sclera, and choroids was also detected (data not shown).

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Figure 4. Cre-recombinase is active in VE-Cadherin-Cre R26R adult endothelium. Adult organs were sectioned on a vibratome and stained for β-gal expression (a,d,g,j,m,p). Stained specimens were subsequently embedded in paraffin, sectioned at 5–7 μ and counterstained with nuclear fast red. a: Heart. b: Histological section of heart. c: High magnification of the heart with β-gal expression in the endothelium of heart vessels. d: Kidney. e: Kidney histology demonstrating LacZ-positive arterial endothelium. f: Capillaries in glomerulus (G) are LacZ positive. g: Brain (pons region). h: Histological section of the brain in g. i: High magnification of brain. Arrows point to β-gal-expressing vessels. j–l: Vibratome and histological sections of lung demonstrate LacZ-positive endothelial cells of alveolar capillaries and large vessels. l: Note that the bronchi (B) are negative. m: Intestine. n: Histological section of the intestine. o: High magnification of intestine reveals LacZ-positive capillary endothelial cells. p: Transversal section of the uterus. q: Whole-mount of the retina. r: Histological section of tongue. s: Histological section of testis. t: Whole-mount of mesentery. u: Histological section of pancreas.

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Reporter expression in blood cells was noted in about half of all circulating cells and in hematopoietic organs, including fetal liver, spleen, thymus, and bone marrow (Fig. 5). β-gal staining in the adult liver was restricted to the veins and sinusoidal vessels (Fig. 5c). LacZ was significantly diminished in the hematopoietic components of adult spleen and thymus (Fig. 5). Nonetheless, LacZ positive cells were strongly noted in the central arteries of the white pulp (Fig. 5g,h) and in the vessels of the thymus (Fig. 5k,l). Interestingly, in adult bone marrow approximately 50% of all hematopoietic lineages were positive for LacZ. A more detailed analysis of this population using both this mouse and a tamoxifen-inducible version of VE-Cadherin-Cre is in preparation (Zovein and colleagues, unpublished results).

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Figure 5. VE-Cadherin drives expression of Cre-recombinase in embryonic and adult hematopoietic organs. a: E14.5 thoracic region depicting liver (L), diaphragm (D), and lung (l). b: High magnification of E14.5 liver. Blood cells in the liver express LacZ. c: Adult liver. d: High magnification of adult liver shows LacZ-positive portal vein (PV) endothelial cells. Arrows point to positive endothelial cells. e: E14.5 spleen (S) and pancreas (P). f: High magnification of E14.5 spleen demonstrates LacZ-positive capillaries and putative hematopoietic cells. Arrows point to vascular expression. g: Vibratome section of adult spleen. h: High magnification of adult spleen. Arrow points to LacZ-positive endothelial cells. i: Vibratome section of E14.5 thymus (T). j: High magnification of E14.5 thymus depicts positive endothelial (arrow) and putative hematopoietic cells (arrowheads). k: Histology of adult thymus. l: High magnification of adult thymus demonstrates positive endothelial cells of a large vessel (arrow). m: Histological section of adult bone marrow spread. n: High magnification of bone marrow reveals positive hematopoietic cells. o,p: Outer cortical bone was sectioned away to allow staining of underlying marrow in whole-mount preparation. o: Adult bone whole-mount. p: High magnification of LacZ-positive marrow within cortical bone.

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Because of its role in cell–cell adhesion, VE-Cadherin has traditionally been considered a marker of fully differentiated endothelial cells. However, work by Nishikawa and coworkers has indicated that, at least early in development, VE-Cadherin labels progenitor cells with the potential to give rise to hematopoietic cells (Hirashima et al.,1999; Nishikawa et al.,1998). More recent reports have indicated that VE-Cadherin expression might be associated with endothelial cell progenitors, fetal liver hematopoietic cells, and embryonic cardiac stem cells (Kim et al.,2005; Iida et al.,2005). Consequently, the activity of the VE-Cadherin promoter fragment in our transgenic Cre mouse appears to recapitulate the expression of the endogenous promoter to include fully differentiated endothelial cells and a subset of hematopoietic cells. These findings reinforce the link between the endothelial and hematopoietic lineages.

Additional analysis of the VE-Cadherin promoter has been recently described in the literature. In particular, the use of 4 kb of regulatory sequences located 5′ of the first intron enhanced reporter expression level in three assay systems including transgenic lines (Hisatsune et al.,2005). The authors were particularly motivated to pursue this investigation because they were unsatisfied with the level of VE-Cadherin-driven reporter expression in brain endothelial cells using the 2.5-kb fragment. This does not appear to be the case in the present Cre-transgenic line, which shows expression in brain capillaries (Fig. 4g–i). Thus, insertion sites might be a possible explanation for the differences between the transgenic lines. Alternatively, β-galactosidase is a more sensitive reporter than CAT or GFP. Based on results from Hisatsune and colleagues, it is likely that a larger promoter fragment might provide stronger expression of Cre-recombinase, yet the levels obtained in this mouse appear to be sufficient to mediate Cre-driven recombination. Interestingly, using the 4-kb fragment, these authors also found that a subset of hematopoietic cells were positive, supporting once again the notion that expression of VE-Cadherin occurs in a population of progenitors with capability to give rise to both endothelium and hematopoietic cells (Hisatsune et al.,2005).

Embryonic stem cell differentiation studies have proposed that smooth muscle cells may also be derived from a common endothelial-hematopoietic progenitor (Yamashita et al.,2000). While this may be the case, lineage analysis using the VE-Cadherin-Cre mouse did not show β-gal expression in smooth muscle cells. This contrasts with a recently reported Flk-1-Cre mouse that revealed expression in muscle cell lineages (Motoike et al.,2003). Much like the Tie-1 and 2 Cre lines previously described (Gustadfsson et al.,2001; Kisanuki et al.,2001), the VE-Cadherin-Cre transgene is restricted to a progenitor capable of differentiation into endothelial or hematopoietic fates, providing further demonstration that Tie-1/2 and VE-Cadherin are developmentally downstream of VEGFR2 (Flk-1). It should be stressed, however, that we have not fully explored the potential fate of these cells under pathological settings. For example, mature endothelial cells exposed to certain stress conditions have been shown to transdifferentiate into cardiac muscle (Condorelli et al.,2001). The VE-Cadherin mouse model offers an excellent genetically traceable system to further examine questions related to transdifferentiation. Our analysis has been restricted to physiological settings and under these conditions we found that transdifferentiation of endothelial cells into other lineages is an unlikely possibility with the exception of the cardiac cushions (Fig. 6). In adult cardiac valves, the contribution of endothelial cell derivatives to putative fibroblasts is significant (Fig. 6e,f). These findings also support those from the Tie-2-Cre mouse (Kisanuki et al.,2001) and provide the opportunity to specifically delete genes within valve-derived cells without affecting fibroblasts at other sites.

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Figure 6. Tracing of endothelial–mesenchymal transition in the cardiac cushions. a: Transversal section of E9.5 heart. b: Higher magnification of a showing positive endocardial cells of the outflow track (black arrow). Note that during this stage, there are also negative cells (arrowhead). β-gal was detected in mesenchymal-like cells (red arrow). c: Transversal histological section of E10.5 heart (A, Atrium; V, Ventricle). Cre activity was detected in most of the endocardial cell population (arrow). Some negative cells are visible mostly in the atrium (arrowhead). d: Higher magnification of c demonstrating positive endocardial cells (black arrows) and positive mesenchymal cells (red arrows). e: Whole-mount section of an adult heart with valves (arrows). f: Histological section of adult heart valve. Arrows denote positive valvular fibroblasts.

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To date five promoters have been used to develop transgenic lines with the objective to target Cre expression to the endothelial cell compartment. Full evaluation of these lines revealed important advantages and some disadvantages. Both Tie-1 and 2 showed early endothelial expression, but demonstrated some limitations. Tie-1 exhibited expression in hematopoietic cells and within some neuronal populations in the cortex and hippocampus (Gustafsson et al.,2001). Tie-2-Cre, the most used line for excision in the endothelium, has also shown expression in the mesoderm (Kisanuki et al.,2001) and both Tie lines expressed Cre in hematopoietic lineages. One of the Flk-1-Cre lines, developed by targeting the Flk-1 locus, demonstrated expression in muscle lineages (Motoike et al.,2003). A second Flk-1-Cre line, utilizing a restricted fragment of the promoter and intron 1, eliminated muscle expression, but was not fully penetrant in quiescent endothelium. The VE-Cadherin-Cre line described here exhibits uniform expression in the endothelium of developing and quiescent vessels of all organs examined. However, it is also expressed within a small compartment of hematopoietic cells and thus analysis of gene deletion should consider this caveat. Although an exclusive endothelial specific line with constitutive expression in embryonic and adult endothelium is yet to be developed, the VE-Cadherin-Cre model adds to previously characterized lines due to its uniform penetration and expression in quiescent endothelium. This is a distinct advantage that may prove important in the functional exploration of physiological and pathological conditions within adult endothelium. Furthermore, this is the first Cre model with demonstrated expression in lymphatic endothelial cells.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Generation of VE-Cadherin-Cre-Recombinase Transgenic Mouse

The VE-Cadherin-Cre construct was generated by inserting the Cre-recombinase cDNA downstream from a 2.5-kb fragment of the VE-Cadherin mouse promoter (a gift from Dr. P. Huber; CEA-INSERM-Joseph Fourier University, France). The transgenic construct was injected into fertilized oocytes from FVB/N mice followed by transfer to foster mothers. Offspring were assessed for Cre-recombinase expression by Southern analysis. Five founders were identified but only three consistently passed the transgene to the next generations. Three lines showed identical patterns of expression. The VE-Cadherin-Cre transgenic mice were bred to the ROSA26R reporter mouse (Jackson Laboratories) to evaluate the activity of the Cre-recombinase. Animals were genotyped by PCR using tail or yolk sac DNA. The presence of the Cre transgene was detected using the following primers: Cre F: 5′GAA CCT GAT GGA CAT GTT CAG GGA and Cre R: 5′ CAG AGT CAT CCT TAG CGC CGT AAA. Genotyping of the Rosa allele was performed as described in Soriano (1999).

Embryonic Dissociation and Cell Staining

Embryos at specific developmental stages (E10.5 and E12.5) were dissected from the placenta and placed in DMEM containing 1 mg/ml collagenase (Sigma, St. Louis, MO). The yolk sacs were used for genotype assessment. Cell dissociation of embryos was accomplished by incubation in the collagenase solution at 37°C for 15 min followed by mechanical disruption (pipetting up and down). Cell suspensions were spun in 1.5-ml eppendorf tubes at 2,000 rpm for 5 min. Supernatant was removed and 1 ml of 1× Ammonium Chloride lysis solution was added and cells incubated for 3 min at room temperature. Embryonic cells were re-pelleted and suspended in 200 μl HBSS buffer (HBSS, 2% FCS, 1% PenStrep, 10 mM HEPES).

Flow Cytometry Analysis

Cells were analyzed and sorted on a FACSAria using PE (phycoerythtin)-conjugated CD41, APC (allophycocyanin)-conjugated PECAM, and 7-AAD (7-amino-actinomycin D) for viability. All monoclonal antibodies, their appropriate isotype controls, and 7-AAD were purchased from BD Pharmingen. Cells were gated based on cell size, viability, and PECAM expression in the absence of CD41 expression with appropriate comparisons to isotype controls. The exclusion of the CD41-positive population would ensure the analysis of an endothelial population that is not contaminated with hematopoietic progenitors, also positive for PECAM. After sorting, cells were stained for β-gal, counterstained with nuclear fast red, and counted under a 40× objective by two independent observers.

Whole-Mount LacZ Staining of Embryos and Histological Analysis

Timed matings were performed to obtain embryos between the stages E6.5 and E17.5 for analysis of LacZ protein expression. Dissected embryos were fixed for 20 min at room temperature in a solution containing 0.2% glutaraldehyde, 5 mM EGTA, 2 mM MgCl2 in phosphate buffered saline (PBS), pH 7.3. Embryos were then rinsed three times for 30 min each in a solution containing 2 mM MgCl2, 0.01% DOC, and 0.02% NP-40, in PBS, pH 7.3. The embryos were stained at 37°C in a solution containing 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% DOC, and 0.02% NP-40, in PBS, pH 7.3. Before staining, embryos E13.5 and older were sectioned on a vibratome at 500–800 μ. For staining of adult organs, mice were initially perfused for 1 min with the fixative solution and organs were then sectioned at 300–600 μ also on a vibratome. Sections were subsequently post-fixed for 10 min, rinsed, and then stained with X-gal. For histological analysis, whole mount embryos and/or vibratomed specimens were embedded in paraffin, sectioned at 5–10 μ and counterstained with nuclear fast red. For evaluation of lymphatic vessels, cryosections (10 μ) of X-gal stained embryos were immunostained with Prox-1 antibody (Covance, 1:5,000) and detection was done using a biotinylated goat anti-rabbit antibody (Jackson Immunoresearch Laboratories, 1:250) as previously described (Wigle and Oliver,1999). The VE-Cadherin-Cre-GFP embryos were evaluated using a confocal microscope. Deparaffinized sections of Xgal stained embryos were incubated with rat anti-mouse CD31 (BD Pharmingen) 1:100, secondary antibody of biotinylated rabbit anti-rat IgG 1:100, and ABC Elite with NovaRED peroxidase stain (Vector laboratories).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank Megan Benedict and Liman Zhao for assistance with animal husbandry and Miriam Dillard for excellent technical work. We also thank the contribution of the FACS Core and the Tissue Procurement Core Laboratory Shared Resource at UCLA. The project was supported, in part, by the National Institutes of Health grant NLHBI HL074455. Additional funding included the following fellowships: Howard Hughes Medical Institute pre-doctoral fellowship to J.A.; NICHD Fellowship, Pediatric Scientist Development Program (NICHD Grant Award K120HD00850) to A.Z.; and Established Investigator of AHA award to L.I.A.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
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