Keiko Kitajima

Epiblast Ground State Is Controlled by Canonical Wnt/β-Catenin Signaling in the Postimplantation Mouse Embryo and Epiblast Stem Cells

Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.

DOCK180 Is a Rac Activator That Regulates Cardiovascular Development by Acting Downstream of CXCR4

Rationale: During embryogenesis, the CXC chemokine ligand (CXCL)12 acts on endothelial cells to control cardiac development and angiogenesis. Although biological functions of CXCL12 are exerted in part through activation of the small GTPase Rac, the pathway leading from its receptor CXC chemokine receptor (CXCR)4 to Rac activation remains to be determined. Objective: DOCK180 (dedicator of cytokinesis), an atypical Rac activator, has been implicated in various cellular functions. Here, we examined the role of DOCK180 in cardiovascular development. Methods and Results: DOCK180 associates with ELMO (engulfment and cell motility) through the N-terminal region containing a Src homology 3 domain. We found that targeted deletion of the Src homology 3 domain of DOCK180 in mice leads to embryonic lethality with marked reduction of DOCK180 expression at the protein level. These mutant mice, as well as DOCK180-deficient mice, exhibited multiple cardiovascular abnormalities resembling those seen in CXCR4-deficient mice. In DOCK180 knocked down endothelial cells, CXCL12-induced Rac activation was impaired, resulting in a marked reduction of cell motility. Conclusions: These results suggest that DOCK180 links CXCR4 signaling to Rac activation to control endothelial cell migration during cardiovascular development.

Reversal of left-right asymmetry induced by aberrant Nodal signaling in the node of mouse embryos

The node at the anterior tip of the primitive streak serves as an initial generator of the left-right (L-R) axis in mammalian embryos. We now show that a small disturbance in molecular signaling at the node is responsible for the L-R reversal of visceral organs in the inv mutant mouse. In the node of wild-type embryos, the expression of Nodal and Cerl2 (Dand5), which encodes an inhibitor of Nodal, is asymmetric, with the level of Nodal expression being higher on the left side and that of Cerl2 expression higher on the right. In inv/inv embryos, however, a localized reduction in the level of Cerl2 expression results in upregulation of the Nodal signal and a consequent induction of Lefty expression in the node. The ectopic expression of Lefty1 delays the onset of Nodal expression in the lateral plate mesoderm. L-R asymmetry of Cerl2 expression in the node also becomes reversed in a manner dependent on the Nodal signal. Nodal expression in the lateral plate mesoderm then appears on the right side, probably reflecting the balance between Nodal and Cerl2 in the node. The inhibition of Cerl2 expression by the Nodal signal suggests a mechanism for amplification of the cue for L-R asymmetry provided by nodal flow and for stabilization of asymmetric gene expression around the node. In inv/inv embryos, this system may function in reverse as a result of ectopic production of Lefty, which inhibits the Nodal signal on the left side in a manner dependent on leftward nodal flow.

Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins

GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)‐hydroxyheptadeca‐5Z,8E,10E‐trienoic acid (12‐HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2‐overexpressing Madin‐Darby canine kidney (MDCK) II cells and in the small intestine of BLT2‐transgenic mice. BLT2‐deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK‐BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK‐Mock cells, and 12‐HHT stimulation accelerated TER recovery only in MDCK‐BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12‐HHT/BLT2 axis up‐regulated claudin‐4 (CLDN4) expression in MDCK‐BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12‐HHT‐dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12‐HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12‐HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein‐p38 MAPK pathway.—Ishii, Y., Saeki, K., Liu, M., Sasaki, F., Koga, T., Kitajima, K., Meno, C., Okuno, T., Yokomizo, T. Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins. FASEB J. 30, 933–947 (2016). www.fasebj.org

Wnt signaling regulates left-right axis formation in the node of mouse embryos.

The node triggers formation of the left-right axis in mouse embryos by establishing local asymmetry of Nodal and Cerl2 expression. We found that Wnt3 is expressed in perinodal crown cells preferentially on the left side. The enhancer responsible for Wnt3 expression was identified and found to be regulated by Foxa2 and Rbpj under the control of Notch signaling. Rbpj binding sites suppress enhancer activity in pit cells of the node, thereby ensuring crown cell-specific expression. In addition, we found that the expression of Gdf1 and Cerl2 is also regulated by Notch signaling, suggesting that such signaling may induce the expression of genes related to left-right asymmetry as a set. Furthermore, Cerl2 expression became symmetric in response to inhibition of Wnt-β-catenin signaling. Our results suggest that Wnt signaling regulates the asymmetry of Cerl2 expression, which likely generates a left-right difference in Nodal activity at the node for further amplification in lateral plate mesoderm.

Restriction of Wnt signaling in the dorsal otocyst determines semicircular canal formation in the mouse embryo

The mouse inner ear develops from a simple epithelial pouch, the otocyst, with the dorsal and ventral portions giving rise to the vestibule and cochlea, respectively. The otocyst undergoes a morphological change to generate flattened saclike structures, known as outpocketings, in the dorsal and lateral regions. The semicircular canals of the vestibule form from the periphery of the outpocketings, with the central region (the fusion plate) undergoing de-epithelialization and disappearing. However, little is known of the mechanism that orchestrates formation of the semicircular canals. We now show that the area of canonical Wnt signaling changes dynamically in the dorsal otocyst during its morphogenesis. The genes for several Wnt ligands were found to be expressed in the dorsal otocyst according to specific patterns, whereas those for secreted inhibitors of Wnt ligands were expressed exclusively in the ventral otocyst. With the use of whole-embryo culture in combination with potent modulators of canonical Wnt signaling, we found that forced persistence of such signaling resulted in impaired formation both of the lateral outpocketing and of the fusion plates of the dorsal outpocketing. Canonical Wnt signaling was found to suppress Netrin1 expression and to preserve the integrity of the outpocketing epithelium. In addition, inhibition of canonical Wnt signaling reduced the size of the otocyst, likely through suppression of cell proliferation and promotion of apoptosis. Our stage-specific functional analysis suggests that strict regulation of canonical Wnt signaling in the dorsal otocyst orchestrates the process of semicircular canal formation.

Dissecting the Role of Fgf Signaling During Gastrulation and Left-Right Axis Formation in Mouse Embryos Using Chemical Inhibitors

Fgf signaling plays pivotal roles in mouse gastrulation and left‐right axis formation. However, although genetic analyses have revealed important aspects of Fgf signaling in these processes, the temporal resolution of genetic studies is low. Here, we combined whole‐embryo culture with application of chemical compounds to inhibit Fgf signaling at specific time points. We found that sodium chlorate and PD173074 are potent inhibitors of Fgf signaling in early mouse embryos. Fgf signaling is required for the epithelial‐to‐mesenchymal transition of the primitive streak before the onset of gastrulation. Once gastrulation begins, Fgf signaling specifies mesodermal fates via the Ras/MAPK downstream cascade. Finally, Fgf signaling on the posterior side of the embryo during gastrulation induces Nodal expression in the node via Tbx6‐Dll1, the initial event required for Nodal expression in the left lateral plate mesoderm. Developmental Dynamics 239:1768–1778, 2010.

Hyperglycemia impairs left-right axis formation and thereby disturbs heart morphogenesis in mouse embryos.

Significance Epidemiological studies have revealed that pregestational diabetes mellitus increases the risk for congenital anomalies, including congenital heart defects (CHDs). Despite the importance of preventing diabetes-related congenital malformations, however, the underlying pathogenic mechanisms have remained largely unknown. Pregestational diabetes mellitus is associated specifically with CHDs accompanied by heterotaxia. We have now examined left–right (L–R) axis formation in embryos of diabetic female mice as well as in mouse embryos exposed to high-glucose concentrations in culture. We found that high-glucose levels prevent establishment of the L–R axis required for heart morphogenesis and the L–R asymmetry of visceral organs. Such a mechanism may thus explain, at least in part, the CHDs and accompanying heterotaxia in the offspring of diabetic mothers. Congenital heart defects with heterotaxia are associated with pregestational diabetes mellitus. To provide insight into the mechanisms underlying such diabetes-related heart defects, we examined the effects of high-glucose concentrations on formation of the left–right axis in mouse embryos. Expression of Pitx2, which plays a key role in left–right asymmetric morphogenesis and cardiac development, was lost in the left lateral plate mesoderm of embryos of diabetic dams. Embryos exposed to high-glucose concentrations in culture also failed to express Nodal and Pitx2 in the left lateral plate mesoderm. The distribution of phosphorylated Smad2 revealed that Nodal activity in the node was attenuated, accounting for the failure of left–right axis formation. Consistent with this notion, Notch signal-dependent expression of Nodal-related genes in the node was also down-regulated in association with a reduced level of Notch signaling, suggesting that high-glucose concentrations impede Notch signaling and thereby hinder establishment of the left–right axis required for heart morphogenesis.

Isolated ACTH deficiency probably induced by autoimmune-related mechanism evoked with nivolumab

Nivolumab, an anti-programmed death-1 antibody, is a breakthrough treatment for several malignancies. Its specific adverse effects caused by autoimmunity are termed immune-related adverse events, which involve several endocrine dysfunctions. Herein, we report two cases of isolated adrenocorticotropic hormone (ACTH) deficiency induced by nivolumab for the treatment of metastatic malignant melanoma. Case 1 was a 39-year-old man and Case 2 was a 50-year-old woman, both of whom presented with progressive melanoma. After 13 courses of nivolumab administration, both cases were diagnosed with adrenal insufficiency. Despite their basal serum ACTH and cortisol levels being low with little response to corticotropin-releasing hormone loading, other anterior pituitary hormone levels were preserved. Based on these endocrinological data, isolated ACTH deficiency was diagnosed. Magnetic resonance imaging showed normal pituitary glands, excluding hypophysitis. Finally, hydrocortisone replacement enabled the patients to continue nivolumab treatment. Therefore, it is important to consider isolated ACTH syndrome as a possible and potentially severe immune-related adverse event of nivolumab, even when head magnetic resonance imaging of affected cases does not show enlargement. We should not misdiagnose hidden immune-related adverse events behind general complaints of malignancies such as general malaise and appetite loss, to allow successful treatment using this beneficial immune checkpoint inhibitor.