Multiphase coalescence mediates Hippo pathway activation.

The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inhibit STRIPAK function. These findings provide a paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a conceptual framework for understanding upstream regulation of the Hippo signaling pathway.

YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.

The Hippo-YAP signaling pathway plays a critical role in development, homeostasis, regeneration, and tumorigenesis by converging on YAP, a coactivator for the TEAD family DNA-binding transcription factors, to regulate downstream transcription programs. Given its pivotal role as the nuclear effector of the Hippo pathway, YAP is indispensable in multiple developmental and tissue contexts. Here we report that the essentiality of YAP in liver and lung development can be genetically bypassed by simultaneous inactivation of the TEAD corepressor VGLL4. This striking antagonistic epistasis suggests that the major physiological function of YAP is to antagonize VGLL4. We further show that the YAP-VGLL4 antagonism plays a widespread role in regulating Hippo pathway output beyond normal development, as inactivation of Vgll4 dramatically enhanced intrahepatic cholangiocarcinoma formation in Nf2-deficient livers and ameliorated CCl4-induced damage in normal livers. Interestingly, Vgll4 expression is temporally regulated in development and regeneration and, in certain contexts, provides a better indication of overall Hippo pathway output than YAP phosphorylation. Together, these findings highlight the central importance of VGLL4-mediated transcriptional repression in Hippo pathway regulation and inform potential strategies to modulate Hippo signaling in cancer and regenerative medicine.

Development of Korea Neuroethics Guidelines.

BACKGROUND: Advances in neuroscience and neurotechnology provide great benefits to humans though unknown challenges may arise. We should address these challenges using new standards as well as existing ones. Novel standards should include ethical, legal, and social aspects which would be appropriate for advancing neuroscience and technology. Therefore, the Korea Neuroethics Guidelines were developed by stakeholders related to neuroscience and neurotechnology, including experts, policy makers, and the public in the Republic of Korea. METHOD: The guidelines were drafted by neuroethics experts, were disclosed at a public hearing, and were subsequently revised by opinions of various stakeholders. RESULTS: The guidelines are composed of twelve issues; humanity or human dignity, individual personality and identity, social justice, safety, sociocultural prejudice and public communication, misuse of technology, responsibility for the use of neuroscience and technology, specificity according to the purpose of using neurotechnology, autonomy, privacy and personal information, research, and enhancement. CONCLUSION: Although the guidelines may require a more detailed discussion after future advances in neuroscience and technology or changes in socio-cultural milieu, the development of the Korea Neuroethics Guidelines is a milestone for the scientific community and society in general for the ongoing development in neuroscience and neurotechnology.

Homeostatic control of Hippo signaling activity revealed by an endogenous activating mutation in YAP.

The Hippo signaling pathway converges on YAP to regulate growth, differentiation, and regeneration. Previous studies with overexpressed proteins have shown that YAP is phosphorylated by its upstream kinase, Lats1/2, on multiple sites, including an evolutionarily conserved 14-3-3-binding site whose phosphorylation is believed to inhibit YAP by excluding it from the nucleus. Indeed, nuclear localization of YAP or decreased YAP phosphorylation at this site (S168 in Drosophila, S127 in humans, and S112 in mice) is widely used in current literature as a surrogate of YAP activation even though the physiological importance of this phosphorylation event in regulating endogenous YAP activity has not been defined. Here we address this question by introducing a Yap(S112A) knock-in mutation in the endogenous Yap locus. The Yap(S112A) mice are surprisingly normal despite nuclear localization of the mutant YAP protein in vivo and profound defects in cytoplasmic translocation in vitro. Interestingly, the mutant Yap(S112A) mice show a compensatory decrease in YAP protein levels due to increased phosphorylation at a mammalian-specific phosphodegron site on YAP. These findings reveal a robust homeostatic mechanism that maintains physiological levels of YAP activity and caution against the assumptive use of YAP localization alone as a surrogate of YAP activity.

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs.

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a "wrapper" around the notochord to constrain these cells along the vertebral column.

Multiphasic and tissue-specific roles of sonic hedgehog in cloacal septation and external genitalia development.

Malformations of the external genitalia are among the most common congenital anomalies in humans. The urogenital and anorectal sinuses develop from the embryonic cloaca, and the penis and clitoris develop from the genital tubercle. Within the genital tubercle, the endodermally derived urethral epithelium functions as an organizer and expresses sonic hedgehog (Shh). Shh knockout mice lack external genitalia and have a persistent cloaca. This identified an early requirement for Shh, but precluded analysis of its later role in the genital tubercle. We conducted temporally controlled deletions of Shh and report that Shh is required continuously through the onset of sexual differentiation. Shh function is divisible into two temporal phases; an anogenital phase, during which Shh regulates outgrowth and patterning of the genital tubercle and septation of the cloaca, and a later external genital phase, during which Shh regulates urethral tube closure. Disruption of Shh function during the anogenital phase causes coordinated anorectal and genitourinary malformations, whereas inactivation during the external genital phase causes hypospadias. Shh directs cloacal septation by promoting cell proliferation in adjacent urorectal septum mesenchyme. Additionally, conditional inactivation of smoothened in the genital ectoderm and cloacal/urethral endoderm shows that the ectoderm is a direct target of Shh and is required for urethral tube closure, highlighting a novel role for genital ectoderm in urethragenesis. Identification of the stages during which disruption of Shh results in either isolated or coordinated malformations of anorectal and external genital organs provides a new tool for investigating the etiology of anogenital malformations in humans.

In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth.

The apical ectodermal ridge (AER) in the vertebrate limb is required for limb outgrowth and patterning. To investigate the role BMP ligands expressed in the AER play in limb development we selectively inactivated both Bmp2 and Bmp4 in this tissue. The autopods of mice lacking both of these genes contained extra digits, digit bifurcations and interdigital webbing due to a decrease in programmed cell death and an increase in cell proliferation in the underlying mesoderm. Upon removal of Bmp2 and Bmp4 in the AER, no defects in proximal-distal patterning were observed. At the molecular level, removal of Bmp2 and Bmp4 in the AER caused an increase in Fgf expression, which correlated with an increase in both the width and length of the AER. Investigation of Engrailed-1 (En1) expression in the AER of limb buds in which Bmp2 and Bmp4 had been removed indicated that En1 expression was absent from this tissue. Our data suggests that AER expression of Bmp2 and Bmp4 is required for digit and dorsal-ventral patterning but surprisingly not for limb outgrowth.

Development of dietary pattern evaluation tool for adults and correlation with Dietary Quality Index.

BACKGROUND/OBJECTIVES: As the prevalence of chronic diseases has risen, the need for straightforward diagnostic tools for monitoring nutrition status to improve nutrition counseling and disease prevention has likewise increased. This study developed an easily usable dietary behavior pattern diagnosis checklist and investigated its correlation with dietary quality index. SUBJECTS/METHODS: A draft dietary pattern evaluation tool was generated by analyzing previous studies. The draft questionnaire comprised 61 questions for assessing dietary habits. A survey was administered to 320 adults (19 to 64 years old) using the dietary pattern evaluation tool and 24-hour-recall method between March and May of 2014 in Jeonbuk province and the metropolitan area. Principal component analysis with varimax rotation was performed to identify dietary behavior patterns. Nutritional analysis was conducted using CAN-Pro 4.0, and the Diet Quality Index-International (DQI-I) was calculated to assess dietary quality. The correlation between dietary pattern scores and DQI-I scores was also analyzed. RESULTS: The factor analysis resulted in a total of 34 questions mapped to four main dietary behavior patterns: "high fat and calorie" pattern (12 questions), "overeating/binge" pattern (nine questions), "dietary impulse" pattern (eight questions), and "unbalanced food intake" pattern (five questions). The four dietary behavior patterns were negatively correlated with DQI-I adequacy and total scores (P < 0.01). CONCLUSIONS: The dietary pattern evaluation tool developed in this study can be used to diagnose a client's dietary behavior problems and is available as a nutrition counseling tool in the field.

The development of a taste education program for preschoolers and evaluation of a program by parents and childcare personnel.

The change in people's dietary life has led to an increase in an intake of processed foods and food chemicals, raising awareness about taste education for preschoolers whose dietary habits start to grow. This study aims to evaluate the effectiveness and satisfaction of parents and childcare personnel after developing a taste education program and demonstrating it in class. A part of the curriculum developed by Piusais and Pierre was referred for the program. After educating 524 preschoolers in child care facilities in Seoul, a satisfaction survey was conducted on the program. The data in this study were analyzed using SPSS 14.0. Statistical analysis was conducted based on the frequency after collecting the data. Mean ± SD used to determine satisfaction with taste education, with preferences marked on a five-point scale and the alpha was set at 0.05. The program includes five teachers' guides with subjects of sweetness, saltiness, sourness, bitterness and harmony of flavor, and ten kinds of teaching tools. For the change in parents' recognition of the need for taste education based on five-point scale, the average of 4.06 ± 0.62 before the program has significantly increased to 4.32 ± 0.52 (P < 0.01). Regarding the change in the preferences for sweetness, saltiness, sourness, and bitterness, the average has increased to 3.83 ± 0.61, 3.62 ± 0.66, 3.64 ± 0.66, and 3.56 ± 0.75 respectively. In an evaluation of instructors in child care facilities, the average scores for education method, education effect, education contents and nutritionists, and teaching tools were at 4.15 ± 0.63, 3.91 ± 0.50, 4.18 ± 0.50, and 3.80 ± 0.56 respectively. In addition, the need for a continuous taste education scored 4.42 ± 0.67. This program has created a positive change in preschoolers' dietary life, therefore the continuation and propagation of the taste education program should be considered.

Sonic hedgehog in the notochord is sufficient for patterning of the intervertebral discs.

The intervertebral discs, located between adjacent vertebrae, are required for stability of the spine and distributing mechanical load throughout the vertebral column. All cell types located in the middle regions of the discs, called nuclei pulposi, are derived from the embryonic notochord. Recently, it was shown that the hedgehog signaling pathway plays an essential role during formation of nuclei pulposi. However, during the time that nuclei pulposi are forming, Shh is expressed in both the notochord and the nearby floor plate. To determine the source of SHH protein sufficient for formation of nuclei pulposi we removed Shh from either the floor plate or the notochord using tamoxifen-inducible Cre alleles. Removal of Shh from the floor plate resulted in phenotypically normal intervertebral discs, indicating that Shh expression in this tissue is not required for disc patterning. In addition, embryos that lacked Shh in the floor plate had normal vertebral columns, demonstrating that Shh expression in the notochord is sufficient for pattering the entire vertebral column. Removal of Shh from the notochord resulted in the absence of Shh in the floor plate, loss of intervertebral discs and vertebral structures. These data indicate that Shh expression in the notochord is sufficient for patterning of the intervertebral discs and the vertebral column.

Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth.

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.

Degeneration and regeneration of the intervertebral disc: lessons from development.

Degeneration of the intervertebral discs, a process characterized by a cascade of cellular, biochemical, structural and functional changes, is strongly implicated as a cause of low back pain. Current treatment strategies for disc degeneration typically address the symptoms of low back pain without treating the underlying cause or restoring mechanical function. A more in-depth understanding of disc degeneration, as well as opportunities for therapeutic intervention, can be obtained by considering aspects of intervertebral disc development. Development of the intervertebral disc involves the coalescence of several different cell types through highly orchestrated and complex molecular interactions. The resulting structures must function synergistically in an environment that is subjected to continuous mechanical perturbation throughout the life of an individual. Early postnatal changes, including altered cellularity, vascular regression and altered extracellular matrix composition, might set the disc on a slow course towards symptomatic degeneration. In this Perspective, we review the pathogenesis and treatment of intervertebral disc degeneration in the context of disc development. Within this scope, we examine how model systems have advanced our understanding of embryonic morphogenesis and associated molecular signaling pathways, in addition to the postnatal changes to the cellular, nutritional and mechanical microenvironment. We also discuss the current status of biological therapeutic strategies that promote disc regeneration and repair, and how lessons from development might provide clues for their refinement.

Relationship between inflammation biomarkers, antioxidant vitamins, and bone mineral density in patients with metabolic syndrome.

Few studies have shown the correlation between metabolic syndrome and bone mineral density (BMD). The main pathogenic mechanisms of metabolic syndrome rely on chronic low-level inflammatory status and oxidative stress. There are few studies that examine the gender-specific effects of inflammation and antioxidants on BMD. In this study, we evaluated the relative contribution of these factors in patients with metabolic syndrome. We conducted a cross-sectional study of 67 men and 46 postmenopausal women with metabolic syndrome; metabolic syndrome was defined as having three or more metabolic syndrome risk factors. BMD, body fat mass, and lean body mass were evaluated. We also examined the levels of high sensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), adiponectin, vitamin E, and C in serum. Log-transformed hs-CRP levels were significantly higher in lumbar spine osteoporotic subjects than in normal subjects for women but not for men. There was no significant difference between the normal group and the osteoporotic group in other inflammatory markers. Stepwise regression analyses for BMD of the lumbar spine showed that lean body mass and vitamin E were significant determinants in men. Lean body mass and log-transformed hs-CRP were significant determinants in women Analysis for BMD of the femoral neck showed that lean body mass was a significant determinant for both men and women. There was no significant factor among the inflammatory markers or antioxidant vitamins affecting the femoral neck BMD for either gender. In conclusion, while hs-CRP is an independent predictor of the BMD of the lumbar spine in women, vitamin E showed profound effects on BMD in men but not women with metabolic syndrome.

Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation.

A classically identified "notochordal" cell population in the nucleus pulposus is thought to regulate disk homeostasis. However, the embryonic origin of these cells has been under dispute for >60 years. Here we provide the first direct evidence that all cell types in the adult mouse nucleus pulposus are derived from the embryonic notochord. Additionally, rare isolated embryonic notochord cells remained in the vertebral column and resembled "notochordal remnants," which in humans have been proposed to give rise to a rare type of late-onset cancer called chordoma. Previously, this cell type had not been identified in the mouse model system. The development and characterization of a mouse model that can be used to fate map nucleus pulposus precursor cells in any mutant background will be useful for uncovering the cellular and molecular mechanisms of disk degeneration. In addition, the identification of notochordal remnants in mice is the first step towards generating an in vivo model of chordoma.