Human epiblast lumenogenesis: From a cell aggregate to a lumenal cyst.

The formation of a central lumen in the human epiblast is a critical step for development. However, because the lumen forms in the epiblast coincident with implantation, the molecular and cellular events of this early lumenogenesis process cannot be studied in vivo. Recent developments using new model systems have revealed insight into the underpinnings of epiblast formation. To provide an up-to-date comprehensive review of human epiblast lumenogenesis, we highlight recent findings from human and mouse models with an emphasis on new molecular understanding of a newly described apicosome compartment, a novel 'formative' state of pluripotency that coordinates with epiblast polarization, and new evidence about the physical and polarized trafficking mechanisms contributing to lumenogenesis.

Functions of TGIF homeodomain proteins and their roles in normal brain development and holoprosencephaly.

Holoprosencephaly (HPE) is a frequent human forebrain developmental disorder with both genetic and environmental causes. Multiple loci have been associated with HPE in humans, and potential causative genes at 14 of these loci have been identified. Although TGIF1 (originally TGIF, for Thymine Guanine-Interacting Factor) is among the most frequently screened genes in HPE patients, an understanding of how mutations in this gene contribute to the pathogenesis of HPE has remained elusive. However, mouse models based on loss of function of Tgif1, and the related Tgif2 gene, have shed some light on how human TGIF1 variants might cause HPE. Functional analyses of TGIF proteins and of TGIF1 single nucleotide variants from HPE patients, combined with analysis of forebrain development in mouse embryos lacking both Tgif1 and Tgif2, suggest that TGIFs regulate the transforming growth factor ß/Nodal signaling pathway and sonic hedgehog (SHH) signaling independently. Although, some developmental processes that are regulated by TGIFs may be Nodal-dependent, it appears that the forebrain patterning defects and HPE in Tgif mutant mouse embryos is primarily due to altered signaling via the Shh pathway.

Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche.

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine.

Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway.

Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh null embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor ß/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway.

Thoracic spinal epidural hematoma misdiagnosed as conversion paralysis: A case report.

A woman with a history of psychiatric hospitalization was misdiagnosed with conversion paralysis despite lower extremity paralysis due to a thoracic epidural hematoma, leaving her with severe neurological deficits. Conversion paralysis is a diagnosis of exclusion and should never be made unless all possible organic causes have been ruled out.

Temporally resolved single cell transcriptomics in a human model of amniogenesis.

Amniogenesis is triggered in a collection of pluripotent epiblast cells as the human embryo implants. To gain insights into the critical but poorly understood transcriptional machinery governing amnion fate determination, we examined the evolving transcriptome of a developing human pluripotent stem cell-derived amnion model at the single cell level. This analysis revealed several continuous amniotic fate progressing states with state-specific markers, which include a previously unrecognized CLDN10+ amnion progenitor state. Strikingly, we found that expression of CLDN10 is restricted to the amnion-epiblast boundary region in the human post-implantation amniotic sac model as well as in a peri-gastrula cynomolgus macaque embryo, bolstering the growing notion that, at this stage, the amnion-epiblast boundary is a site of active amniogenesis. Bioinformatic analysis of published primate peri-gastrula single cell sequencing data further confirmed that CLDN10 is expressed in cells progressing to amnion. Additionally, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses primordial germ cell-like fate. Overall, this study presents a comprehensive amniogenic single cell transcriptomic resource and identifies a previously unrecognized CLDN10+ amnion progenitor population at the amnion-epiblast boundary of the primate peri-gastrula.

Impact of renal denervation on quality of life (How does renal denervation contribute to improving hypertension treatment affected by poor medication adherence?).

The US Food and Drug Administration has approved renal denervation (RDN) as a new treatment option for hypertension (HT) because it not only has antihypertensive effects but also improves the quality of blood pressure (BP) reduction. RDN is expected to be increasingly used in clinical practice in the future. This review summarizes the impact of RDN on quality of life (QOL). Although the treatment of HT aims to improve life prognosis, the use of antihypertensive agents can impair QOL because of adverse effects and lifestyle changes associated with long-term medication use. Consequently, poor adherence to antihypertensive agents is a common problem and may be the most important issue affecting patient QOL. In RDN trials in patients taking antihypertensive agents, approximately 40% of patients had poor adherence to the drugs. Poor adherence is often the cause of resistant hypertension. Therefore, RDN should be well suited to treating HT and improving QOL. Studies have shown that approximately 30% of HT patients prefer RDN to drug treatment. Patients who prefer RDN are typically male and younger and have high BP, poor adherence, and a history of adverse effects of antihypertensive agents. We hope that RDN will improve not only life prognosis but also QOL in HT patients because of its benefits for adherence. Furthermore, we expect that in the future, RDN will be used in other sympathetic nervous system-related diseases, such as heart failure, atrial fibrillation, and sleep apnea syndrome.

Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification.

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that bone morphogenetic protein (BMP) signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hr after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification.

Temporally resolved early BMP-driven transcriptional cascade during human amnion specification.

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that BMP signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hours after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification.

Tgif1 and Tgif2 regulate Nodal signaling and are required for gastrulation.

Tgif1 and Tgif2 are transcriptional co-repressors that limit the response to TGFbeta signaling and play a role in regulating retinoic-acid-mediated gene expression. Mutations in human TGIF1 are associated with holoprosencephaly, but it is unclear whether this is a result of deregulation of TGFbeta/Nodal signaling, or of effects on other pathways. Surprisingly, mutation of Tgif1 in mice results in only relatively mild developmental phenotypes in most strain backgrounds. Here, we show that loss-of-function mutations in both Tgif1 and Tgif2 result in a failure of gastrulation. By conditionally deleting Tgif1 in the epiblast, we demonstrate that a single wild-type allele of Tgif1 in the extra-embryonic tissue allows the double null embryos to gastrulate and begin organogenesis, suggesting that extra-embryonic Tgif function is required for patterning the epiblast. Genetically reducing the dose of Nodal in embryos lacking all Tgif function results in partial rescue of the gastrulation defects. Conditional double null embryos have defects in left-right asymmetry, which are also alleviated by reducing the dose of Nodal. Together, these data show that Tgif function is required for gastrulation, and provide the first clear evidence that Tgifs limit the transcriptional response to Nodal signaling during early embryogenesis.

Monitoring Effects of Membrane Traffic Via Changes in Cell Polarity and Morphogenesis in Three-Dimensional Human Pluripotent Stem Cell Cysts.

Membrane traffic at the Golgi and endosomes plays many critical roles in the polarization and the morphogenesis of epithelial tissues. Studies into the roles of traffic in morphogenesis in mammals are often complicated by early embryonic lethality of mutations in membrane traffic as well as the inherent difficulty in imaging developing embryos posed by their size and location. Increasingly, human pluripotent stem cell (hPSC)-derived embryo- and organ-like systems (e.g., embryoids, organoids) provide a useful platform to illuminate the requirements of traffic in human embryonic tissue morphogenesis because these in vitro models are highly amenable to fluorescence microscopy and provide the ability to examine the role of essential genes not possible with animal studies. Here, we present a method to generate hPSC-cysts, a 3-D hPSC-based model of human epiblast lumen formation. This system provides unique opportunities to examine the role of membrane traffic during epithelial morphogenesis. We also present methods to process hPSC-cysts for immunofluorescence and staining with commonly used fluorescence labels useful for detecting defects in polarization and morphogenesis caused by defects in membrane traffic.

The mammalian formin FHOD1 is activated through phosphorylation by ROCK and mediates thrombin-induced stress fibre formation in endothelial cells.

Formin-family proteins, in the active state, form actin-based structures such as stress fibres. Their activation mechanisms, however, are largely unknown except that mDia and its closely related formins can be activated by direct binding of the small GTPase Rho or Cdc42. Here we show that the Rho-dependent protein kinase ROCK phosphorylates the C-terminal residues Ser1131, Ser1137, and Thr1141 of formin homology domain protein 1 (FHOD1), a major endothelial formin that is normally autoinhibited by intramolecular interaction between the N- and C-terminal regions. Phosphorylation of FHOD1 at the three residues fully disrupts the autoinhibitory interaction, which culminates in formation of stress fibres. We also demonstrate that, in vascular endothelial cells, thrombin, a vasoactive substance leading to Rho activation, elicits both FHOD1 phosphorylation and stress fibre formation in a ROCK-dependent manner, and that FHOD1 depletion by RNA interference impairs thrombin-induced stress fibre formation. Based on these findings we propose a novel mechanism for activation of formin-family proteins: ROCK, activated by G protein-coupled receptor ligands such as thrombin, directly phosphorylates FHOD1 at the C-terminal region, which renders this formin in the active form, leading to stress fibre formation.

[Evaluation of preoperative intraaortic balloon pumping for high risk off-pump coronary artery bypass grafting].

We evaluated the effect of preoperative intraaortic balloon pumping (IABP) support in high risk patients undergoing off-pump coronary artery bypass grafting (OPCAB). Between November 1999 and December 2010, 65 high-risk patients underwent OPCAB with the support of IABP inserted preoperatively. High risks were considered as (1) left main coronary artery stem stenosis > or = 75%, (2) unstable angina requiring intravenous nitrates and heparin, (3) preoperative left ventricular ejection fraction < or = 30%, (4) bilateral carotid artery stenosis > or = 75%. There were no hospital deaths or cerebrovascular complications. During operations, hemodynamics was stable with the support of low dose catecholamines, and no patient needed conversion to on-pump coronary artery bypass grafting. All patients were able to be weaned from IABP within 3 days (mean 5.7 hours) after the operation and were extubated within 4 days (mean 11.5 hours) after the operation. One patient had a peripheral embolism which might be related to insertion of IABP (1.5%). Preoperative IABP in high-risk patients undergoing OPCAB was considered to be useful and safe.

Spatially resolved cell polarity proteomics of a human epiblast model.

Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.

Mammalian formin fhod3 regulates actin assembly and sarcomere organization in striated muscles.

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.

Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells.

Neural rosettes (NPC rosettes) are radially arranged groups of cells surrounding a central lumen that arise stochastically in monolayer cultures of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPC). Since NPC rosette formation is thought to mimic cell behavior in the early neural tube, these rosettes represent important in vitro models for the study of neural tube morphogenesis. However, using current protocols, NPC rosette formation is not synchronized and results are inconsistent among different hPSC lines, hindering quantitative mechanistic analyses and challenging live cell imaging. Here, we report a rapid and robust protocol to induce rosette formation within 6 h after evenly-sized "colonies" of NPC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NPC monolayers. These NPC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL, an important apical determinant recently identified as a candidate gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals that, in addition to radial organization and apical lumen formation, cells within cut NPC colonies initiate rapid basally-driven spreading. Further, using chemical, genetic and biomechanical tools, we show that NPC rosette morphogenesis requires this basal spreading activity and that spreading is tightly regulated by Rho/ROCK signaling. This robust and quantitative NPC rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NPC rosette morphogenesis.

Heterogeneity of Kir4.1 channel expression in glia revealed by mouse transgenesis.

The weakly inwardly rectifying K(+) channel Kir4.1 is found in many glial cells including astrocytes. However, questions remain regarding the relative contribution of Kir4.1 to the resting K(+) conductance of mature astrocytes in situ. We employed a bacterial artificial chromosome transgenic approach in mice to visualize Kir4.1 expression in vivo. These mice (Kir4.1-EGFP) express enhanced green fluorescent protein (EGFP) under the transcriptional control of the Kir4.1 promoter. The brains of adult Kir4.1-EGFP transgenic mice showed co-expression of EGFP and Kir4.1 in astrocytes. In addition, weaker expression of EGFP was detected in NG2+ glial cells when compared with EGFP expression in GFAP+ glial cells. Whole-cell voltage clamp recordings of EGFP+ glial cells in the CA1 area of the adult mouse hippocampus indicated astrocytes displaying properties consistent with both the "passive" and "complex" subpopulations. EGFP+ cells with bright fluorescence had the linear current-voltage (I-V) relationships and extensive gap junctional coupling characteristic of passive astrocytes. However, EGFP+ glia with weaker fluorescence displayed properties associated with complex astrocytes including nonlinear I-V relationships and lack of intercellular gap junctional coupling. Pharmacological blockade of inward currents implied that Kir4.1 channels constitute the dominant resting K(+) conductance in both glial cell types and are more highly expressed in passive astrocytes. These results suggest differential expression of Kir4.1 in glia and that this channel likely underlies the resting K(+) conductance in passive and complex astrocytes.

Opening the black box: Stem cell-based modeling of human post-implantation development.

Proper development of the human embryo following its implantation into the uterine wall is critical for the successful continuation of pregnancy. However, the complex cellular and molecular changes that occur during this post-implantation period of human development are not amenable to study in vivo. Recently, several new embryo-like human pluripotent stem cell (hPSC)-based platforms have emerged, which are beginning to illuminate the current black box state of early human post-implantation biology. In this review, we will discuss how these experimental models are carving a way for understanding novel molecular and cellular mechanisms during early human development.

Genetic and Molecular Analyses indicate independent effects of TGIFs on Nodal and Gli3 in neural tube patterning.

Holoprosencephaly (HPE) is a prevalent craniofacial developmental disorder that has both genetic and environmental causes. The gene encoding TG-interacting factor 1 (TGIF1) is among those that are routinely screened in HPE patients. However, the mechanisms by which TGIF1 variants cause HPE are not fully understood. TGIF1 is a transcriptional repressor that limits the output of the Transforming Growth Factor ß (TGFß)/Nodal signaling pathway, and HPE in patients with TGIF1 variants has been suggested to be due to increased Nodal signaling. Mice lacking both Tgif1 and its paralog, Tgif2, have HPE, and embryos lacking Tgif function do not survive past mid-gestation. Here, we show that in the presence of a Nodal heterozygous mutation, proliferation defects are rescued and a proportion of embryos lacking all Tgif function survive to late gestation. However, these embryos have a classic HPE phenotype, suggesting that this is a Nodal-independent effect of Tgif loss of function. Further, we show that the Gli3 gene is a direct target for repression by Tgifs, independent of TGFß/Nodal signaling, consistent with Tgif mutations causing HPE via Nodal-independent effects on the Sonic Hedgehog (Shh) pathway. Based on this work, we propose a model for distinct functions of Tgifs in the Nodal and Shh/Gli3 pathways during forebrain development.