Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.

Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility.

Obesity and diabetes-related kidney diseases associate with renal failure and cardiovascular morbidity, and represent a major health issue worldwide. However, the molecular mechanisms leading to their development remain poorly understood. We observed increased expression of transcription factor FoxC2 in the podocytes of obese Zucker rats that are insulin resistant and albuminuric. We also found that depletion of adiponectin, an adipocyte-derived hormone whose secretion is decreased in obesity, upregulated FOXC2 in differentiated human podocytes in vitro. Overexpression of FOXC2 in cultured human podocytes led to increased nuclear expression of FOXC2 associated with a change of cellular morphology. This was accompanied by upregulation of vimentin, a key mesenchymal marker, and active beta-catenin, associated with podocyte injury. We also observed re-organization of the actin cytoskeleton, disrupted localization of the tight junction protein ZO-1, and increased motility of podocytes overexpressing FOXC2. These data indicate that the expression of FOXC2 in podocytes needs to be tightly regulated, and that its overexpression induces a chain of cellular events leading to podocyte dysfunction. These changes may lead to podocyte detachment and depletion ultimately contributing to albuminuria. We also suggest a novel molecular mechanism linking obesity-induced decrease in adiponectin to podocyte dysfunction via upregulation of FOXC2.

Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias.

AIM: Ventricular fibrillation (VF), the main cause of sudden cardiac death (SCD), occurs most frequently in the acute phase of myocardial infarction: a certain fraction of VF, however, develops in an apparently healthy heart, referred as idiopathic VF. The contribution of perturbation in the fast conduction system in the ventricle, the His-Purkinje system, for idiopathic VF has been implicated, but the underlying mechanism remains unknown. Irx3/IRX3 encodes a transcription factor specifically expressed in the His-Purkinje system in the heart. Genetic deletion of Irx3 provides a mouse model of ventricular fast conduction disturbance without anatomical or contraction abnormalities. The aim of this study was to examine the link between perturbed His-Purkinje system and idiopathic VF in Irx3-null mice, and to search for IRX3 genetic defects in idiopathic VF patients in human. METHODS AND RESULTS: Telemetry electrocardiogram recording showed that Irx3-deleted mice developed frequent ventricular tachyarrhythmias mostly at night. Ventricular tachyarrhythmias were enhanced by exercise and sympathetic nerve activation. In human, the sequence analysis of IRX3 exons in 130 probands of idiopathic VF without SCN5A mutations revealed two novel IRX3 mutations, 1262G>C (R421P) and 1453C>A (P485T). Ventricular fibrillation associated with physical activities in both probands with IRX3 mutations. In HL-1 cells and neonatal mouse ventricular myocytes, IRX3 transfection up-regulated SCN5A and connexin-40 mRNA, which was attenuated by IRX3 mutations. CONCLUSION: IRX3 genetic defects and resultant functional perturbation in the His-Purkinje system are novel genetic risk factors of idiopathic VF, and would improve risk stratification and preventive therapy for SCD in otherwise healthy hearts.

Control of retinoid levels by CYP26B1 is important for lymphatic vascular development in the mouse embryo.

During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.

Effects of Hydroxy Groups in the A-Ring on the Anti-proteasome Activity of Flavone.

The ubiquitin-proteasome pathway plays an important role in regulating apoptosis and the cell cycle. Recently, proteasome inhibitors have been shown to have antitumor effects and have been used in anticancer therapy for several cancers such as multiple myeloma. Although some flavones, such as apigenin, chrysin and luteolin, have a specific role in the inhibition of proteasome activity and induced apoptosis in some reports, these findings did not address all flavone types. To further investigate the proteasome-inhibitory mechanism of flavonoids, we examined the inhibitory activity of 5,6,7-trihydroxyflavone, baicalein and 5,6,7,4'-tetrahydroxyflavone, scutellarein on extracted proteasomes from mice and cancer cells. Unlike the other flavones, baicalein and scutellarein did not inhibit proteasome activity or accumulate levels of ubiquitinated proteins. These results indicate that flavones with hydroxy groups at positions 5, 6 and 7 of the A-ring lack the anti-proteasome function.

Generation and characterization of Tbx1-AmCyan1 transgenic reporter mouse line that selectively labels developing thymus primordium.

Thymus development is a complicated process that includes highly dynamic morphological changes and reciprocal tissue interactions between endoderm-derived epithelial cells of the anterior foregut and neural crest-derived mesenchymal cells. We generated and characterized a Tbx1-AmCyan1 reporter transgenic mouse to visualize thymus precursor cells during early embryonic development. In transgenic embryos, AmCyan1 fluorescence was specifically detected in the endoderm of the developing 3rd and 4th pharyngeal pouches and later in thymus epithelium until E14.5. Cells expressing AmCyan1 that were isolated based on AmCyan1 fluorescence expressed endodermal, thymic, and parathyroid markers, but they did not express neural crest or endothelial markers; these findings indicated that this transgenic mouse strain could be used to collect thymic or parathyroid precursor cells or both. We also showed that in nude mice, which exhibit defects in thymus development, the thymus precursors were clearly labeled with AmCyan1. In summary, these AmCyan1-fluorescent transgenic mice are useful for investigating early thymus development.

Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome.

The regulation of endogenous retinoic acid level through CYP26B1 is required for elevation of palatal shelves.

BACKGROUND: In previous studies, we investigated the effects of excess retinoic acid (RA) during palatogenesis by RA administration to pregnant mice. In the present study, we deleted Cyp26b1, one of the RA-degrading enzymes, to further study the effects of excess RA in the normal developing palate and to understand how endogenous levels of RA are regulated. RESULTS: Excess RA, due to the absence of Cyp26b1, targets cells in the bend region of the palatal shelves and inhibits their horizontal elevation, leading to cleft palate. An organ culture of Cyp26b1-/- palatal shelves after tongue removal did not rescue the impaired elevation of the palatal shelves. The expression of Fgf10, Bmp2, and Tbx1, important molecules in palatal development, was down-regulated. Cell proliferation was decreased in the bend region of palatal shelves. Tongue muscles were hypoplastic and/or missing in Cyp26b1-/- mice. CONCLUSIONS: We demonstrated that CYP26B1 is essential during palatogenesis. Excess RA due to the lack of Cyp26b1 suppresses the expression of key regulators of palate development in the bend region, resulting in a failure in the horizontal elevation of the palatal shelves. The regulation of RA signaling through CYP26B1 is also necessary for the development of tongue musculature and for tongue depression.

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes.

The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with ∼50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development.

Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development.

Identification of targets of Prox1 during in vitro vascular differentiation from embryonic stem cells: functional roles of HoxD8 in lymphangiogenesis.

During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.

Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis.

Lymphatic vessels are essential for the removal of interstitial fluid and prevention of tissue edema. Lymphatic capillaries lack associated mural cells, and collecting lymphatic vessels have valves, which prevent lymph backflow. In lymphedema-distichiasis (LD), lymphatic vessel function fails because of mutations affecting the forkhead transcription factor FOXC2. We report that Foxc2(-/-) mice show abnormal lymphatic vascular patterning, increased pericyte investment of lymphatic vessels, agenesis of valves and lymphatic dysfunction. In addition, an abnormally large proportion of skin lymphatic vessels was covered with smooth muscle cells in individuals with LD and in mice heterozygous for Foxc2 and for the gene encoding lymphatic endothelial receptor, Vegfr3 (also known as Flt4). Our data show that Foxc2 is essential for the morphogenesis of lymphatic valves and the establishment of a pericyte-free lymphatic capillary network and that it cooperates with Vegfr3 in the latter process. Our results indicate that an abnormal interaction between the lymphatic endothelial cells and pericytes, as well as valve defects, underlie the pathogenesis of LD.

Carcinoma cells that have undergone an epithelial-mesenchymal transition differentiate into endothelial cells and contribute to tumor growth.

Hypoxia stimulates neoangiogenesis, promoting tumor outgrowth, and triggers the epithelial-mesenchymal transition (EMT), which bestows cells with mesenchymal traits and multi-lineage differentiation potential. Here, we investigated whether EMT can confer endothelial attributes upon carcinoma cells, augmenting tumor growth and vascularization. Following orthotopic implantation of MCF-7 human epithelial breast cancer cells into mice, tumors of different sizes were immunostained for markers of hypoxia and EMT. Larger tumors were well-vascularized with CD31-positive cells of human origin. Hypoxic regions, demarcated by HIF-1α staining, exhibited focal areas of E-cadherin loss and elevated levels of vimentin and the EMT-mediator FOXC2. Implantation of MCF-7 cells, co-mixed with human mammary epithelial (HMLE) cells overexpressing the EMT-inducer Snail, markedly potentiated tumor growth and vascularization, compared with MCF-7 cells injected alone or co-mixed with HMLE-vector cells. Intra-tumoral vessels contained CD31-positive cells derived from either donor cell type. FOXC2 knockdown abrogated the potentiating effects of HMLE-Snail cells on MCF-7 tumor growth and vascularization, and compromised endothelial transdifferentiation of mesenchymal cells cultured in endothelial growth medium. Hence, cells that have undergone EMT can promote tumor growth and neovascularization either indirectly, by promoting endothelial transdifferentiation of carcinoma cells, or directly, by acquiring an endothelial phenotype, with FOXC2 playing key roles in these processes.

A unique expression pattern of Tbx10 in the hindbrain as revealed by Tbx10(LacZ) allele.

To study the expression/function of Tbx10, a T-box gene, Tbx10(LacZ/+) mice were established by replacing the T-box coding region with a LacZ gene. X-gal staining showed that LacZ(+) cells were localized to two-cell populations in rhombomere 4 and rhombomere 6. No significant differences in the locations of LacZ(+) cells were found between Tbx10(LacZ/+) and Tbx10(LacZ/LacZ) mice, and the Tbx10(LacZ/LacZ) mice were viable and fertile. We found that the LacZ(+) cells are present in both embryonic and adult mice. Histological studies suggest that the rhombomere 4-derived LacZ(+) cells are a subpopulation of the ventral interneurons in the pons.

Organ-specific lymphangiectasia, arrested lymphatic sprouting, and maturation defects resulting from gene-targeting of the PI3K regulatory isoforms p85alpha, p55alpha, and p50alpha.

The phosphoinositide 3-kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here, we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85alpha, p55alpha, and p50alpha impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage-dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell-origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up-regulation of the transforming growth factor-beta co-receptor endoglin, and reduced levels of mature vascular endothelial growth factor-C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis.

Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow.

The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome.

The medaka FoxP2, a homologue of human language gene FOXP2, has a diverged structure and function.

Forkhead box (Fox) genes are involved in organogenesis and cell differentiation. A mutation of FOXP2 was discovered in patients with severe defects in speech and language. The medaka FoxP2 was cloned in order to clarify the molecular evolution and difference in the protein structure and function by comparing human/mouse and medaka genes. The result showed that medaka FoxP2 had a 73.7% homology to the human and mouse counterparts, and its zinc finger, leucine zipper and forkhead domain structures were conserved. However, medaka FoxP2 lacked a long polyglutamine repeat and had two insertions of unique amino acid sequences. FoxP2 expression was found in the epiphysis and retina, in addition to the midbrain and cerebellum. The transcriptional assay revealed that medaka FoxP2 showed a very weak repressive activity to the CC10 promoter while mouse Foxp2 exhibited a strong repressive activity. Mutational analyses of medaka FoxP2 showed that the three amino acids of forkhead domain were responsible for the weak repressive activity. These results suggest that medaka FoxP2 may play a different function in the development of the medaka fish.

Foxc2CreERT2 knock-in mice mark stage-specific Foxc2-expressing cells during mouse organogenesis.

Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2CreERT2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2CreERT2 ; R26R), the fate of Foxc2 positive (Foxc2+ ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2+ cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2+ descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2+ cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2+ cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2CreERT2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2+ expressing cells during mouse embryogenesis.

Roles of forkhead transcription factor Foxc2 (MFH-1) and endothelin receptor A in cardiovascular morphogenesis.

OBJECTIVE: Foxc2/MFH-1 is a member of the forkhead family of transcription factors and Foxc2-deficient mice exhibit aortic arch anomalies (type B interruption of the aortic arch). Endothelin receptor type-A (ETA) is one of the two known endothelin receptors that belong to the G-protein-coupled receptor family. ETA-deficient mice show defects in the great arteries, primarily type B interruption of the aortic arch. Based on similar phenotypes in the cardiovascular system of Foxc2- and ETA-deficient mice, we investigated whether Foxc2 and ETA have a close relationship in aortic arch patterning. METHODS: The Foxc2 and ETA homozygotes were obtained by crossing the Foxc2 and ETA heterozygotes, respectively. The double Foxc2/ETA homozygotes were obtained by crossing the double Foxc2/ETA heterozygotes. RESULTS: We investigated the expression of ETA in Foxc2-null mice and the expression of Foxc2 in ETA-null mice and found that the absence of either Foxc2 or ETA had no effect on the expression of the other. Next, we analyzed mice lacking both Foxc2 and ETA to examine the relationship between Foxc2 and ETA on aortic arch patterning in vivo. We found that the majority of Foxc2/ETA double-mutant embryos died around 11.5 dpc and that all surviving mice had persistent truncus arteriosus. CONCLUSIONS: The results suggest that Foxc2- and ETA-expressing cells additively form the aorticopulmonary septum.

Data supporting the regulation of FOXC2 in podocyte dysfunction.

This data article shows the expression levels of specific podocyte injury markers and podocyte slit diaphragm protein nephrin in obese and lean Zucker rat glomeruli. It also contains information on the effect of the overexpression of transcription factor FOXC2 on the ratio of F- and G-actin and the expression level of ZO-1 in differentiated human podocytes. The article also shows data on the effect of treatments of differentiated podocytes with various factors associated with obesity and diabetes on the expression level of FOXC2. The detailed interpretation of these data and other aspects of podocyte injury mediated by upregulation of FOXC2 can be found in "Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility [1].