Gastrulation morphogenesis in synthetic systems.

Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures, entirely in vitro. This recreation of embryonic form from naïve cells, known as synthetic morphogenesis, has important implications for both developmental biology and regenerative medicine. However, the capacity of stem cell-based models to recapitulate the morphogenetic cell behaviors that shape natural embryos remains unclear. In this review, we explore several examples of synthetic morphogenesis, with a focus on models of gastrulation and surrounding stages. By varying cell types, source species, and culture conditions, researchers have recreated aspects of primitive streak formation, emergence and elongation of the primary embryonic axis, neural tube closure, and more. Here, we describe cell behaviors within in vitro/ex vivo systems that mimic in vivo morphogenesis and highlight opportunities for more complete models of early development.

Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.

Vertebrate heart development requires spatiotemporal regulation of gene expression to specify cardiomyocytes, increase the cardiomyocyte population through proliferation, and to establish and maintain atrial and ventricular cardiac chamber identities. The evolutionarily conserved chromatin factor Gon4-like (Gon4l), encoded by the zebrafish ugly duckling (udu) locus, has previously been implicated in cell proliferation, cell survival, and specification of mesoderm-derived tissues including blood and somites, but its role in heart formation has not been studied. Here we report two distinct roles of Gon4l/Udu in heart development: regulation of cell proliferation and maintenance of ventricular identity. We show that zygotic loss of udu expression causes a significant reduction in cardiomyocyte number at one day post fertilization that becomes exacerbated during later development. We present evidence that the cardiomyocyte deficiency in udu mutants results from reduced cell proliferation, unlike hematopoietic deficiencies attributed to TP53-dependent apoptosis. We also demonstrate that expression of the G1/S-phase cell cycle regulator, cyclin E2 (ccne2), is reduced in udu mutant hearts, and that the Gon4l protein associates with regulatory regions of the ccne2 gene during early embryogenesis. Furthermore, udu mutant hearts exhibit a decrease in the proportion of ventricular cardiomyocytes compared to atrial cardiomyocytes, concomitant with progressive reduction of nkx2.5 expression. We further demonstrate that udu and nkx2.5 interact to maintain the proportion of ventricular cardiomyocytes during development. However, we find that ectopic expression of nkx2.5 is not sufficient to restore ventricular chamber identity suggesting that Gon4l regulates cardiac chamber patterning via multiple pathways. Together, our findings define a novel role for zygotically-expressed Gon4l in coordinating cardiomyocyte proliferation and chamber identity maintenance during cardiac development.

Capital juror questionnaires in death-penalty cases: A study of attitudes, denials, and deceptions.

The Sixth Amendment right to an "impartial jury" should guarantee fundamental fairness that in capital cases may literally be a matter of life and death. For ecological validity, the current study focuses on capital jury questionnaires (CJQs) employed in actual death-penalty cases. Study I examined 248 undergraduates and their responses to death-penalty relevant questions. As an MTurk investigation, Study II consisted of 259 community members potentially eligible for capital trial jury trials. Misrepresentations were operationalized as either denials (concealing their true views) or outright deceptions (dissembling the opposite viewpoint). Both studies found that CJQ items were very susceptible to both types of misrepresentation, irrespective of support-life or support-death views. Nearly 30% of undergraduates openly acknowledged that they would misrepresent close to half their CJQ responses. Overall, community members were much more willing to engage in denials and outright deceptions. The discussion focuses on how CJQs could be improved to promote candor about death-penalty views.

Faking Good: An Investigation of Social Desirability and Defensiveness in an Inpatient Sample With Personality Disorder Traits.

Accurate interpretations of psychological assessments rely heavily on forthright reporting. However, researchers and practitioners recognize that examinees can easily invalidate their test results by underreporting symptoms or overstating positive attributes. Rogers (2008) delineated two distinct but related forms of positive impression management (PIM): defensiveness (denying symptoms and psychological impairment) and social desirability (putting forth an exaggeratedly positive image). Although these two have often been combined in past research, this study sought to investigate each separately via a mixed within- and between-subjects simulation design. Simulation scenarios included a special rehabilitation program for the defensiveness (DF) condition and a competitive job for social desirability (SD). The study used the Personality Inventory for DSM-5 (PID-5; Krueger, Derringer, Markon, Watson, & Skodol, 2012) and recruited 106 inpatients from a psychiatric hospital. As expected, inpatients with prominent personality traits substantially suppressed them under both PIM conditions. Having shown the susceptibility of the PID-5 to intentional distortion, two empirically derived and conceptually based validity scales were next developed to address this important concern. Pending further validation, they might contribute to screening PIM presentations, thus promoting the PID-5's clinical utility. Continued research is needed across multiscale inventories for differentiating PIM presentations.

One American Perspective on the Rights of Accused: An Initial Survey of Miranda Rights in a Broader Context.

In the wake of countless police dramas, commonly held misperceptions endure that the American public knows both Miranda warnings and concomitant rights. Past research has tested public knowledge of Miranda per se, without evaluating additional misconceptions. The current investigation utilizes the European Union's much more all-encompassing safeguards, as delineated in the EU's 2012 Directive and Letter of Rights. Besides knowledge of Miranda, the advisability of these enhanced rights and protections was also assessed. In order to obtain a cross-section of the community, 619 participants were recruited from actual jury pools. Interestingly, they believed that Miranda afforded arrestees many more protections than it actually does. In general, nearly all (>90%) agreed that the accused should be given accurate information (e.g., charges and alleged criminal acts) coupled with an absence of police deception. The potential implications of these findings are discussed as they relate to police practices and due process. Copyright © 2016 John Wiley & Sons, Ltd.

Temporal dynamics of BMP/Nodal ratio drive tissue-specific gastrulation morphogenesis.

Anteroposterior (AP) elongation of the vertebrate body plan is driven by convergence and extension (C&E) gastrulation movements in both the mesoderm and neuroectoderm, but how or whether molecular regulation of C&E differs between tissues remains an open question. Using a zebrafish explant model of AP axis extension, we show that C&E of the neuroectoderm and mesoderm can be uncoupled ex vivo, and that morphogenesis of individual tissues results from distinct morphogen signaling dynamics. Using precise temporal manipulation of BMP and Nodal signaling, we identify a critical developmental window during which high or low BMP/Nodal ratios induce neuroectoderm- or mesoderm-driven C&E, respectively. Increased BMP activity similarly enhances C&E specifically in the ectoderm of intact zebrafish gastrulae, highlighting the in vivo relevance of our findings. Together, these results demonstrate that temporal dynamics of BMP and Nodal morphogen signaling activate distinct morphogenetic programs governing C&E gastrulation movements within individual tissues.

Rbm8a deficiency causes hematopoietic defects by modulating Wnt/PCP signaling.

Defects in blood development frequently occur among syndromic congenital anomalies. Thrombocytopenia-Absent Radius (TAR) syndrome is a rare congenital condition with reduced platelets (hypomegakaryocytic thrombocytopenia) and forelimb anomalies, concurrent with more variable heart and kidney defects. TAR syndrome associates with hypomorphic gene function for RBM8A/Y14 that encodes a component of the exon junction complex involved in mRNA splicing, transport, and nonsense-mediated decay. How perturbing a general mRNA-processing factor causes the selective TAR Syndrome phenotypes remains unknown. Here, we connect zebrafish rbm8a perturbation to early hematopoietic defects via attenuated non-canonical Wnt/Planar Cell Polarity (PCP) signaling that controls developmental cell re-arrangements. In hypomorphic rbm8a zebrafish, we observe a significant reduction of cd41-positive thrombocytes. rbm8a-mutant zebrafish embryos accumulate mRNAs with individual retained introns, a hallmark of defective nonsense-mediated decay; affected mRNAs include transcripts for non-canonical Wnt/PCP pathway components. We establish that rbm8a-mutant embryos show convergent extension defects and that reduced rbm8a function interacts with perturbations in non-canonical Wnt/PCP pathway genes wnt5b, wnt11f2, fzd7a, and vangl2. Using live-imaging, we found reduced rbm8a function impairs the architecture of the lateral plate mesoderm (LPM) that forms hematopoietic, cardiovascular, kidney, and forelimb skeleton progenitors as affected in TAR Syndrome. Both mutants for rbm8a and for the PCP gene vangl2 feature impaired expression of early hematopoietic/endothelial genes including runx1 and the megakaryocyte regulator gfi1aa. Together, our data propose aberrant LPM patterning and hematopoietic defects as consequence of attenuated non-canonical Wnt/PCP signaling upon reduced rbm8a function. These results also link TAR Syndrome to a potential LPM origin and a developmental mechanism.

Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population.

BACKGROUND: During gastrulation, an embryo acquires the three primordial germ layers that will give rise to all of the tissues in the body. In amniote embryos, this process occurs via an epithelial to mesenchymal transition (EMT) of epiblast cells at the primitive streak. Although the primitive streak is vital to development, many aspects of how it forms and functions remain poorly understood. RESULTS: Using live, 4 dimensional imaging and immunohistochemistry, we have shown that the posterior epiblast of the pre-streak murine embryo does not display convergence and extension behavior or large scale migration or rearrangement of a cell population. Instead, the primitive streak develops in situ and elongates by progressive initiation EMT in the posterior epiblast. Loss of basal lamina (BL) is the first step of this EMT, and is strictly correlated with ingression of nascent mesoderm. Once the BL is lost in a given region, cells leave the epiblast by apical constriction in order to enter the primitive streak. CONCLUSIONS: This is the first description of dynamic cell behavior during primitive streak formation in the mouse embryo, and reveals mechanisms that are quite distinct from those observed in other amniote model systems. Unlike chick and rabbit, the murine primitive streak arises in situ by progressive initiation of EMT beginning in the posterior epiblast, without large-scale movement or convergence and extension of epiblast cells.

Vangl2 deficient zebrafish exhibit hallmarks of neural tube closure defects.

Neural tube defects (NTDs) are among the most devastating and common congenital anomalies worldwide, and the ability to model these conditions in vivo is essential for identifying causative genetic and environmental factors. Although zebrafish are ideal for rapid candidate testing, their neural tubes develop primarily via a solid neural keel rather that the fold-and-fuse method employed by mammals, raising questions about their suitability as an NTD model. Here, we demonstrate that despite outward differences, zebrafish anterior neurulation closely resembles that of mammals. For the first time, we directly observe fusion of the bilateral neural folds to enclose a lumen in zebrafish embryos. The neural folds fuse by zippering between multiple distinct but contiguous closure sites. Embryos lacking vangl2, a core planar cell polarity and NTD risk gene, exhibit delayed neural fold fusion and abnormal neural groove formation, yielding distinct openings and midline bifurcations in the developing neural tube. These data provide direct evidence for fold-and-fuse neurulation in zebrafish and its disruption upon loss of an NTD risk gene, highlighting conservation of vertebrate neurulation and the utility of zebrafish for modeling NTDs.

Cardiac Phenotyping of SARS-CoV-2 in British Columbia: A Prospective Echo Study With Strain Imaging.

BACKGROUND: There is limited data on the residual echocardiographic findings including strain analysis among post-coronavirus disease (COVID) patients. The aim of our study is to prospectively phenotype post-COVID patients. METHODS: All patients discharged following acute COVID infection were systematically followed in the post-COVID-19 Recovery Clinic at Vancouver General Hospital and St. Paul's Hospital. At 4-18 weeks post diagnosis, patients underwent comprehensive echocardiographic assessment. Left ventricular ejection fraction (LVEF) was assessed by 3D, 2D Biplane Simpson's, or visual estimate. LV global longitudinal strain (GLS) was measured using a vendor-independent 2D speckle-tracking software (TomTec). RESULTS: A total of 127 patients (53% female, mean age 58 years) were included in our analyses. At baseline, cardiac conditions were present in 58% of the patients (15% coronary artery disease, 4% heart failure, 44% hypertension, 10% atrial fibrillation) while the remainder were free of cardiac conditions. COVID-19 serious complications were present in 79% of the patients (76% pneumonia, 37% intensive care unit admission, 21% intubation, 1% myocarditis). Normal LVEF was seen in 96% of the cohort and 97% had normal right ventricular systolic function. A high proportion (53%) had abnormal LV GLS defined as < 18%. Average LV GLS of septal and inferior segments were lower compared to that of other segments. Among patients without pre-existing cardiac conditions, LVEF was abnormal in only 1.9%, but LV GLS was abnormal in 46% of the patients. CONCLUSIONS: Most post-COVID patients had normal LVEF at 4-18 weeks post diagnosis, but over half had abnormal LV GLS.

Generation of Naïve Blastoderm Explants from Zebrafish Embryos.

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively naïve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise naïve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Nodal and planar cell polarity signaling cooperate to regulate zebrafish convergence and extension gastrulation movements.

During vertebrate gastrulation, convergence and extension (C and E) of the primary anteroposterior (AP) embryonic axis is driven by polarized mediolateral (ML) cell intercalations and is influenced by AP axial patterning. Nodal signaling is essential for patterning of the AP axis while planar cell polarity (PCP) signaling polarizes cells with respect to this axis, but how these two signaling systems interact during C and E is unclear. We find that the neuroectoderm of Nodal-deficient zebrafish gastrulae exhibits reduced C and E cell behaviors, which require Nodal signaling in both cell- and non-autonomous fashions. PCP signaling is partially active in Nodal-deficient embryos and its inhibition exacerbates their C and E defects. Within otherwise naïve zebrafish blastoderm explants, however, Nodal induces C and E in a largely PCP-dependent manner, arguing that Nodal acts both upstream of and in parallel with PCP during gastrulation to regulate embryonic axis extension cooperatively.

Cellular and molecular mechanisms of convergence and extension in zebrafish.

Gastrulation is the period of development when the three germ layers, mesoderm, endoderm and ectoderm, are not only formed, but also shaped into a rudimentary body plan. An elongated anteroposterior (AP) axis is a key feature of all vertebrate body plans, and it forms during gastrulation through the highly conserved morphogenetic mechanism of convergence & extension (C&E). As the name suggests, this process requires that cells within each germ layer converge toward the dorsal midline to narrow the tissue in the mediolateral (ML) dimension and concomitantly extend it in the AP dimension. In a number of vertebrate species, C&E is driven primarily by mediolateral intercalation behavior (MIB), during which cells elongate, align, and extend protrusions in the ML direction and interdigitate between their neighbors. MIB is only one of many complex cellular mechanisms that contributes to C&E in zebrafish embryos, however, where a combination of individual cell migration, collective migration, random walk, radial intercalation, epiboly movements, and MIB all act together to shape the nascent germ layers. Each of these diverse cell movements is driven by a distinct suite of dynamic cellular properties/activities, such as actin-rich protrusions, myosin contractility, and blebbing. Here, we discuss the spatiotemporal patterns of cellular behaviors underlying C&E gastrulation movements within each germ layer of zebrafish embryos. These behaviors must be coordinated with the embryonic axes, and we highlight the roles of Planar Cell Polarity (PCP) in orienting and BMP signaling in patterning C&E cell behaviors with respect to the AP and dorsoventral axes. Finally, we address the role of GPCR signaling, extracellular matrix, and mechanical signals in coordination of C&E movements between adjacent germ layers.

Malingering and Defensiveness on the Spanish Personality Assessment Inventory: An Initial Investigation with Mostly Spanish-Speaking Outpatients.

Psychological assessments can be essentially invalidated by examinees' intentional response styles, such as feigning (i.e., fabrication or marked overreporting of symptoms/impairment) and defensiveness (i.e., denial or minimization of symptoms/impairment). As a psychometric strength, the Personality Assessment Inventory (PAI) has established validity indicators for identifying both response styles. With the United States' increasing ethnic and cultural diversity, predominantly Spanish-speaking individuals are now estimated in the range of 15 million persons. Unfortunately, very little research has been conducted on the Spanish-translated PAI regarding its effectiveness in clinical populations. Using a between-subjects design, a sample of mostly Spanish-speaking outpatients was randomly assigned to genuine, feigning, or defensive conditions. For feigning, PAI malingering indicators using rare symptoms strategies (i.e., Negative Impression [NIM] and Negative Distortion [NDS] scales) demonstrated moderate to large effect sizes. For defensiveness, the Defensive (DEF) index proved the most effective with a very large effect size (M = 1.68). Different cut scores were examined to increase the clinical utility of the Spanish PAI for determining response styles.

Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes.

Anteroposterior (AP) axis extension during gastrulation requires embryonic patterning and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation through direct positive and negative regulation of gene expression. Although identified as a recessive enhancer of impaired axis extension in planar cell polarity (PCP) mutants, udu functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. Gon4l limits expression of the cell-cell and cell-matrix adhesion molecules EpCAM and Integrinα3b, excesses of which perturb the notochord boundary via tension-dependent and -independent mechanisms, respectively. By promoting formation of this AP-aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis along the AP embryonic axis.

Regulation of gastrulation movements by emergent cell and tissue interactions.

It is during gastrulation that the primordial germ layers are specified, embryonic axes become morphologically manifest, and the embryonic body plan begins to take shape. As morphogenetic movements push and pull nascent tissues into position within the gastrula, new interactions are established between neighboring cells and tissues. These interactions represent an emergent property within gastrulating embryos, and serve to regulate and promote ensuing morphogenesis that establishes the next set of cell/tissue contacts, and so on. Several recent studies demonstrate the critical roles of such interactions during gastrulation, including those between germ layers, along embryonic axes, and at tissue boundaries. Emergent tissue interactions result from - and result in - morphogen signaling, cell contacts, and mechanical forces within the gastrula. Together, these comprise a dynamic and complex regulatory cascade that drives gastrulation morphogenesis.

Evaluating juvenile detainees' Miranda misconceptions: The discriminant validity of the Juvenile Miranda Quiz.

Most juvenile arrestees in custodial settings waive their Miranda rights almost immediately, and many then provide incriminating statements, if not outright confessions. Forensic practitioners are then asked to provide retrospective determinations regarding whether these waivers were effectuated knowingly, voluntarily, and intelligently. At present, the forensic assessment instrument for juvenile Miranda issues consists of the Miranda Rights Comprehension Instruments (MRCI)-which as its name implies-focuses mostly on Miranda comprehension with a de-emphasis of Miranda reasoning. In partially addressing this gap, the current study investigated the clinical utility of the Juvenile Miranda Quiz (JMQ) for evaluating key Miranda misconceptions, a critically important component of Miranda reasoning. Using data from 201 juvenile detainees, we evaluated the JMQ's discriminability with regards to cognitive variables and MRCI scales. Many moderate effect sizes in the predicted direction were found for the JMQ Primary Total and Juvenile Total scores. Finally, these detainees were tested using a mock crime scenario with a representative Miranda warning plus a brief interrogation to evaluate whether they would waive their rights, and if so, whether they would confess. Using Miranda measures to predict problematic outcomes (i.e., impaired waivers followed by confessions), the JMQ Juvenile Total proved the most successful. These findings are discussed within the context of the "intelligent" prong of Miranda waivers. (PsycINFO Database Record

Dodging Self-Incriminations: An Examination of Feigned Miranda Abilities on the SAMA.

Forensic assessments must always consider whether examinees are putting forth genuine effort or seeking to feign legally relevant incapacities. Miranda abilities are no exception when a putatively invalid Miranda waiver might result in the full suppression of an outright confession. Using a within-subjects simulation design, jail detainees were administered a representative Miranda warning and two Standardized Assessment of Miranda Abilities (SAMA) measures: Miranda Vocabulary Scale and Miranda Quiz. As expected, detainees have no difficulty in feigning severe deficits in their recall of the Miranda warning and portraying markedly impaired abilities on both SAMA measures. However, using floor-effect detection strategies, several feigning indicators proved effective at identifying likely feigned Miranda abilities. As an ancillary issue, the Inventory of Legal Knowledge was found to be very effective using both the traditional and revised scoring.

Validity and Representative Data of the MRCI With Legally Involved Juveniles.

Recognized for nearly four decades, most juvenile suspects waive their Miranda rights and almost immediately provide self-incriminating evidence. Miranda-specific measures were eventually developed to understand their capacities and limitations. With extensive revisions, the Miranda Rights Comprehension Instruments (MRCI) were normed and validated. Beyond reliability, the current study addresses the convergent and discriminant validity of the MRCI. In response to Frumkin and Sellbom's criticism of the MRCI's norms, the current research provides representative data on 245 legally involved juveniles with percentiles to facilitate the interpretation of MRCI data. The current investigation is also the first MRCI study to link directly Miranda comprehension (i.e., the knowing prong) to Miranda reasoning (i.e., the intelligent prong) of waiver decisions.