Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines.

The CreERT2/loxP system is widely used to induce conditional gene deletion in mice. One of the main advantages of the system is that Cre-mediated recombination can be controlled in time through Tamoxifen administration. This has allowed researchers to study the function of embryonic lethal genes at later developmental timepoints. In addition, CreERT2 mouse lines are commonly used in combination with reporter genes for lineage tracing and mosaic analysis. In order for these experiments to be reliable, it is crucial that the cell labeling approach only marks the desired cell population and their progeny, as unfaithful expression of reporter genes in other cell types or even unintended labeling of the correct cell population at an undesired time point could lead to wrong conclusions. Here we report that all CreERT2 mouse lines that we have studied exhibit a certain degree of Tamoxifen-independent, basal, Cre activity. Using Ai14 and Ai3, two commonly used fluorescent reporter genes, we show that those basal Cre activity levels are sufficient to label a significant amount of cells in a variety of tissues during embryogenesis, postnatal development and adulthood. This unintended labelling of cells imposes a serious problem for lineage tracing and mosaic analysis experiments. Importantly, however, we find that reporter constructs differ greatly in their susceptibility to basal CreERT2 activity. While Ai14 and Ai3 easily recombine under basal CreERT2 activity levels, mTmG and R26R-EYFP rarely become activated under these conditions and are therefore better suited for cell tracking experiments.

Adgrf5 contributes to patterning of the endothelial deep layer in retina.

Neovascularization of the inner retinal space is a major cause of vision loss. In retinal angiomatous proliferation (RAP) syndrome, newly formed vessels originate from the retinal plexus and invade the inner retinal space. However, the molecular pathways preventing subretinal vascularization remain largely unknown. In most murine models of RAP, pathological neovascularization occurs concomitantly with the development of the retinal vasculature. Here, we demonstrate that disturbing the sequence of morphogenetic events that shape the three-layered retinal vascular network leads to subretinal vascularization. Sprouts emerging from the perivenous region after the first postnatal week extended toward the retinal space where they merged into the deep layer. The small GTPase Rac1 was required for the formation of these vascular extensions and the vascular inner plexus is formed coaxially to the overarching veins. The adhesion receptor Adgrf5 was highly expressed in the endothelium of the central nervous system, where it regulates blood-brain barrier formation. The vascular superficial plexus of Adgrf5 mutant mouse retinae exhibited an increased vascular density in the perivenous areas with increased projections toward the inner plexus where they subsequently created hyper-dense endothelial cells (EC) clusters. Disturbing the perivenous pool of EC thus significantly altered the inner plexus formation. These abnormalities culminated in transient vascular protrusions in the inner retinal space. Taken together, these results reveal a previously unobserved vascular morphogenetic defect in Adgrf5 knockout mice, implicating a role for ADGRF5 in the initiation of subretinal vascularization. Our findings also illustrate how vein-derived EC shape the inner retinal layer formation and could control the appearance of angiomatous malformations.

TGF-ß1-induced EMT promotes targeted migration of breast cancer cells through the lymphatic system by the activation of CCR7/CCL21-mediated chemotaxis.

Tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial-mesenchymal transition (EMT) in response to transforming growth factor-ß (TGF-ß1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF-ß1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF-ß1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF-ß1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF-ß1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells.

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

Nephrin is involved in podocyte maturation but not survival during glomerular development.

Nephrin, a major component of the glomerular slit diaphragm (SD), is both a structural protein as well as a signaling molecule influencing foot process (FP) formation and maintenance of podocyte integrity. Analyses of near-term embryonic kidneys showed normal cellular viability and no apoptosis in glomeruli from nephrin knockout mice. Moreover, expression and location of other SD or glomerular basement membrane components were similar in wild-type and mutant mice as was the location and levels of most podocyte-specific proteins. Transcriptional profiling showed that the lack of nephrin had minor impact on the expression of genes for FPs and SD proteins. Claudin 3, a tight-junction protein normally absent in glomeruli, was upregulated threefold in the knockout mice, suggesting a role of nephrin in claudin 3 gene expression within the glomeruli. Our results suggest that nephrin is expressed late in the process of podocyte differentiation and is a locus for the formation of SD and FP maintenance and physical integrity in vivo. Nephrin does not seem to have a primary role in cell survival but has a small impact on gene regulation during glomerular development.

Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis.

The kidney glomerulus plays a crucial role in blood filtration but the molecular composition and physiology of the glomerulus is not well understood. We previously constructed and large-scale sequenced four mouse glomerular expressed sequence tag (EST) libraries from newborn and adult mouse glomeruli. Here, we compared glomerular EST profiles with whole kidney EST profiles, thereby identifying 497 transcripts corresponding to UniGene clusters that were glomerulus-enriched, that is expressed more abundantly in glomeruli than in whole kidney. These include several known protein-coding glomerulus-specific transcripts critical for glomerulus development and function, but also a large number of gene transcripts, which have not previously been shown to be expressed in the glomerulus, or implicated in glomerular functions. We used in situ hybridization to demonstrate glomerulus-specific RNA expression for six novel glomerular genes and the public Human Protein Atlas to verify glomerular protein expression for another two. The higher mRNA abundance for the eight genes in glomeruli compared with whole kidney was also verified by Taqman quantitative polymerase chain reaction. We surmise that the further characterization of these genes and proteins will increase our understanding of glomerular development and physiology.

Heparan sulphate requirement in platelet-derived growth factor B-mediated pericyte recruitment.

HS (heparan sulphate) plays a key role in angiogenesis, by interacting with growth factors required in the process. It has been proposed that HS controls the diffusion, and thus the availability, of platelet-derived growth factor B that is needed for pericyte recruitment around newly formed capillaries. The present paper summarizes our studies on the importance of HS structure in this regulatory process.

Platelet-derived growth factor B-chain of hematopoietic origin is not necessary for granulation tissue formation and its absence enhances vascularization.

The hypothesis that wound repair is augmented by delivery of platelet-derived growth factor (PDGF) from platelets and macrophages is an attractive extrapolation from the known activities of PDGF in cell culture and in vivo. To test this hypothesis in mice, we prepared hematopoietic chimeras, in which the hematopoietic system of a normal adult mouse was replaced by the hematopoietic system of a PDGF B-chain -/- or +/+ donor. We initiated local granulation tissue formation either by implanting small surgical sponges to elicit a foreign body granulation tissue response, or by ligating the left common carotid to form an organized thrombus. We found that the absence of hematopoietic PDGF B-chain did not decrease the extent of granulation tissue or vascular lesion formation, and that the vascularization of both lesions increased by approximately 100%. We conclude that PDGF B-chain from cells of hematopoietic origin, including platelets and macrophages, is not important for granulation tissue formation, and that it reduces vascularization of granulation issue, probably through disabling of the short-range chemotactic gradients of PDGF that are important for recruiting pericytes/smooth muscle cells to the endothelium of new vessels.

Characterization of a nicotinamide nucleotide transhydrogenase gene from the green alga Acetabularia acetabulum and comparison of its structure with those of the corresponding genes in mouse and Caenorhabditis elegans.

Proton-pumping nicotinamide nucleotide transhydrogenase (Nnt) is a membrane-bound enzyme that catalyzes the reversible reduction of NADP(+) by NADH. This reaction is linked to proton translocation across the membrane. Depending on metabolic conditions, the enzyme may be involved in NADPH generation, e.g., for detoxification of peroxides and/or free radicals and protection from ischemic damage. Nnt exists in most prokaryotes and in animal mitochondria. It is composed of 2-3 subunits in bacteria and of a single polypeptide in mitochondria. An open question is whether Nnt exists in any photosynthetic eukaryotes and if so, to which class it belongs. In the present study it is demonstrated that, by cloning and sequencing cDNA and genomic copies of its NNT gene, an ancient alga, Acetabularia acetabulum (Chlorophyta, Dasycladales), contains a nuclear-encoded Nnt. In contrast to photosynthetic bacteria, this algal Nnt is composed of a single polypeptide of the class found in animal mitochondria. Excluding a poly(A) tail, NNT cDNA from A. acetabulum is 3688 bp long, consists of eight exons and spans 17 kb. The NNT gene from mouse was also characterized. Subsequently, the gene organization of the A. acetabulum NNT was compared to those of the homologous mouse (100 kb and 21 exons) and Caenorhabditis elegans (5.1 kb and 18 exons) genes.

Vascular endothelial growth factor-B-deficient mice display an atrial conduction defect.

BACKGROUND: Vascular endothelial growth factors (VEGFs) and their receptors are essential regulators of vasculogenesis and angiogenesis in both embryos and adults. One of the factors with a still unknown physiological function is VEGF-B, which is expressed in many tissues, including the heart. METHODS AND RESULTS: Mice carrying a targeted deletion in the VEGF-B gene were developed. In VEGF-B(-/-) animals, no gross abnormalities were observed in organs that normally show high expression of VEGF-B, such as the heart, muscle, and kidney. Analysis of heart function by ECG showed that adult VEGF-B(-/-) mice have an atrial conduction abnormality characterized by a prolonged PQ interval. VEGF- or basic fibroblast growth factor-induced corneal angiogenesis was similar in normal and VEGF-B(-/-) mice. CONCLUSIONS: VEGF-B seems to be required for normal heart function in adult animals but is not required for proper development of the cardiovascular system either during development or for angiogenesis in adults.

Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor.

Caffeine is believed to act by blocking adenosine A(1) and A(2A) receptors (A(1)R, A(2A)R), indicating that some A(1) receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A(1)R (A(1)R(-/-)). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A(1)R(+/+) mice, but A(1)R(-/-) mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A(1)R(-/-) mice. In A(1)R(+/-) mice the potency of adenosine was halved, as was the number of A(1)R. In A(1)R(-/-) mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A(1)Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.

Developmental roles of platelet-derived growth factors.

Platelet-derived growth factor (PDGF) was originally identified in platelets and in serum as a mitogen for fibroblasts, smooth muscle cells (SMC) and glia cells in culture. PDGF has since expanded to a family of dimers of at least four gene products, whose biological actions are mediated through two receptor tyrosine kinases, PDGFRs. The present review summarizes and discusses the biological functions of PDGFs and PDGFRs in developmental processes, mainly as revealed through genetic analysis in mice. Such studies have demonstrated multiple critical roles of PDGFs and PDGFRs in embryonic and postnatal development. PDGFs seem to act upon specific populations of progenitor cells that give rise to several different cell types with distinct functions in a variety of developmental processes. Analogies are seen between the cell functions and the developmental processes controlled by PDGFs. This suggests that ancestral PDGF and PDGFR expression patterns and functions may have been iterated in related sets of morphogenetic processes in the course of evolution.

Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis.

The association of pericytes (PCs) to newly formed blood vessels has been suggested to regulate endothelial cell (EC) proliferation, survival, migration, differentiation, and vascular branching. Here, we addressed these issues using PDGF-B-- and PDGF receptor-beta (PDGFR-beta)--deficient mice as in vivo models of brain angiogenesis in the absence of PCs. Quantitative morphological analysis showed that these mutants have normal microvessel density, length, and number of branch points. However, absence of PCs correlates with endothelial hyperplasia, increased capillary diameter, abnormal EC shape and ultrastructure, changed cellular distribution of certain junctional proteins, and morphological signs of increased transendothelial permeability. Brain endothelial hyperplasia was observed already at embryonic day (E) 11.5 and persisted throughout development. From E 13.5, vascular endothelial growth factor-A (VEGF-A) and other genes responsive to metabolic stress became upregulated, suggesting that the abnormal microvessel architecture has systemic metabolic consequences. VEGF-A upregulation correlated temporally with the occurrence of vascular abnormalities in the placenta and dilation of the heart. Thus, although PC deficiency appears to have direct effects on EC number before E 13.5, the subsequent increased VEGF-A levels may further abrogate microvessel architecture, promote vascular permeability, and contribute to formation of the edematous phenotype observed in late gestation PDGF-B and PDGFR-beta knock out embryos.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF beta-receptor null mice.

Platelet-derived growth factor (PDGF)-B and PDGF beta-receptor (PDGFR beta) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B(-/-), PDGFR beta(-/-), or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFR beta expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses. (Blood. 2001;97:1990-1998)

Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis.

Development of the gastrointestinal (GI) tract depends on reciprocal epithelial-mesenchymal cell signaling. Here, we demonstrate a role for platelet-derived growth factor-A (PDGF-A) and its receptor, PDGFR-(alpha), in this process. Mice lacking PDGF-A or PDGFR-(alpha) were found to develop an abnormal GI mucosal lining, including fewer and misshapen villi and loss of pericryptal mesenchyme. Onset of villus morphogenesis correlated with the formation of clusters of PDGFR-(alpha) positive cells, 'villus clusters', which remained located at the tip of the mesenchymal core of the growing villus. Lack of PDGF-A or PDGFR-(alpha) resulted in progressive depletion of PDGFR-(alpha) positive mesenchymal cells, the formation of fewer villus clusters, and premature expression of smooth muscle actin (SMA) in the villus mesenchyme. We found that the villus clusters were postmitotic, expressed BMP-2 and BMP-4, and that their formation correlated with downregulated DNA synthesis in adjacent intestinal epithelium. We propose a model in which villus morphogenesis is initiated as a result of aggregation of PDGFR-(&agr;) positive cells into cell clusters that subsequently function as mesenchymal centers of signaling to the epithelium. The role of PDGF-A seems to be to secure renewal of PDGFR-(alpha) positive cells when they are consumed in the initial rounds of cluster formation.

Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice.

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.

Islet amyloid polypeptide (amylin)-deficient mice develop a more severe form of alloxan-induced diabetes.

To examine whether islet amyloid polypeptide (IAPP), other than through amyloid formation, may be of importance in diabetes pathogenesis, IAPP-deficient mice (IAPP(-/-)) were challenged with alloxan (day 0). Diabetes in IAPP(-/-) mice was more severe at day 35, indicated by greater weight loss; glucose levels were higher in alloxan-treated IAPP(-/-) mice, whereas insulin levels were lower, indicating a greater impairment of islet function. Accordingly, glucose levels upon intravenous glucose challenges at days 7 and 35 were consistently higher in alloxan-treated IAPP(-/-) mice. At day 35, insulin mRNA expression, but not beta-cell mass, was lower in untreated IAPP(-/-) mice. Yet, upon alloxan administration, beta-cell mass and numbers of beta-cell-containing islets were significantly more reduced in IAPP(-/-) mice. Furthermore, they displayed exaggerated beta-cell dysfunction, because in their remaining beta-cells, insulin mRNA expression was significantly more impaired and the localization of glucose transporter-2 was perturbed. Thus the lack of IAPP has allowed exaggerated beta-cell cytotoxic actions of alloxan, suggesting that there may be beneficial features of IAPP actions in situations of beta-cell damage.

Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice.

Mucosal surfaces are protected specifically by secretory immunoglobulin A (SIgA) and SIgM generated through external translocation of locally produced dimeric IgA and pentameric IgM. Their active transport is mediated by the epithelial polymeric Ig receptor (pIgR), also called the transmembrane secretory component. Paracellular passive external transfer of systemic and locally produced antibodies also provides mucosal protection, making the biological importance of secretory immunity difficult to assess. Here we report complete lack of active external IgA and IgM translocation in pIgR knockout mice, indicating no redundancy in epithelial transport mechanisms. The knockout mice were of normal size and fertility but had increased serum IgG levels, including antibodies to Escherichia coli, suggesting undue triggering of systemic immunity. Deterioration of their epithelial barrier function in the absence of SIgA (and SIgM) was further attested to by elevated levels of albumin in their saliva and feces, reflecting leakage of serum proteins. Thus, SIgA did not appear to be essential for health under the antigen exposure conditions of these experimental animals. Nevertheless, our results showed that SIgA contributes to maintenance of mucosal homeostasis. Production of SIgA might therefore be a variable in the initiation of human immunopathology such as inflammatory bowel disease or gluten-sensitive enteropathy.

EPS8 and E3B1 transduce signals from Ras to Rac.

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.