Wataru Kimura

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

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.

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. genesis, 2011.

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. Developmental Dynamics 241:1744–1756, 2012.

Origin of pancreatic precursors in the chick embryo and the mechanism of endoderm regionalization.

To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.

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. Copyright

The mouse forkhead gene Foxp2 modulates expression of the lung genes

AIMS Foxp2 is expressed in the lung during mouse development. A monoclonal anti-mouse Foxp2 antibody was created to determine the expression pattern in the developing lung. Next, transcriptional control of two lung genes, CC10 and surfactant protein C (SPC) genes, by Foxp2 was investigated in H441 and A549 cells. Thirdly, expression patterns of Foxp2 and Foxf2 were compared in the developing lung. Finally, Foxp2 expression was determined in the Foxf2-null mice. MAIN METHODS Immunohistochemical staining and in situ hybridization were applied to the sections of lungs in the developing embryos. KEY FINDINGS Monoclonal anti-Foxp2 antibody demonstrated that Foxp2 was expressed in the bronchial epithelium at E10.5 and its expression became restricted to the distal portion of the elongating bronchiolar epithelium and finally to type II alveolar epithelial cells around birth and in the adult. Foxp2 activated the SPC gene promoter in the presence of Nkx2.1 in A549 cells while it repressed the CC10 gene promoter in H441 cells. Next, the expression domains of the Foxp2 and Foxf2 were found to be exclusive in the lung. Finally, the expression of Foxp2 did not change in the lung of Foxf2-null mice. SIGNIFICANCE The Foxp2 protein is expressed in the growing distal edge of airway epithelium. When the bronchiolus elongates, Foxp2 suppresses CC10 expression. When the lung alveolus is formed, Foxp2 modulates the Nkx2.1-mediated SPC expression in type II alveolar cells. Foxp2 and Foxf2 independently play distinct roles in the alveoli and the mesenchyme, respectively.

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.

A unique expression pattern of Tbx10 in the hindbrain as revealed by Tbx10LacZ allele

To study the expression/function of Tbx10, a T‐box gene, Tbx10LacZ/+ 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 Tbx10LacZ/+ and Tbx10LacZ/LacZ mice, and the Tbx10LacZ/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. genesis, 48:295–302, 2010.

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.