An ethical framework for human embryology with embryo models.

A human embryo's legal definition and its entitlement to protection vary greatly worldwide. Recently, human pluripotent stem cells have been used to form in vitro models of early embryos that have challenged legal definitions and raised questions regarding their usage. In this light, we propose a refined legal definition of an embryo, suggest "tipping points" for when human embryo models could eventually be afforded similar protection to that of embryos, and then revisit basic ethical principles that might help to draft a roadmap for the gradual, justified usage of embryo models in a manner that aims to maximize benefits to society.

Graded mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart.

Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development at single-cell resolution. Nascent mesoderm progenitors form opposing density and motility gradients, converting the temporal birth sequence of gastrulation into a spatial anterolateral-to-posteromedial arrangement. Migrating precardiac mesoderm does not strictly preserve cellular neighbor relationships, and spatial patterns only become solidified as the cardiac crescent emerges. Progenitors undergo a mesenchymal-to-epithelial transition, with a first heart field (FHF) ridge apposing a motile juxta-cardiac field (JCF). Anchored along the ridge, the FHF epithelium rotates the JCF forward to form the initial heart tube, along with push-pull morphodynamics of the second heart field. In Mesp1 mutants that fail to make a cardiac crescent, mesoderm remains highly motile but directionally incoherent, resulting in density gradient inversion. Our practicable live embryo imaging approach defines spatial origins and behaviors of cardiac progenitors and identifies their unanticipated morphological transitions.

Human Embryogenesis: A Comparative Perspective.

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.

A conserved role of the Hippo signalling pathway in initiation of the first lineage specification event across mammals.

Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental programme in mouse, cow and human embryos. However, the mechanisms transducing cell polarity into cell fate in cow and human embryos are unknown. Here, we have examined the evolutionary conservation of Hippo signalling, which is thought to function downstream of aPKC activity, in four different mammalian species: mouse, rat, cow and human. In all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2. However, the timing and localisation of molecular markers differ across species, with rat embryos more closely recapitulating human and cow developmental dynamics, compared with the mouse. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.

Integrative analysis of Lunatic Fringe variants associated with spondylocostal dysostosis type-III.

Lunatic Fringe (LFNG) is required for spinal development. Biallelic pathogenic variants cause spondylocostal dysostosis type-III (SCD3), a rare disease generally characterized by malformed, asymmetrical, and attenuated development of the vertebral column and ribs. However, a variety of SCD3 cases reported have presented with additional features such as auditory alterations and digit abnormalities. There has yet to be a single, comprehensive, functional evaluation of causative LFNG variants and such analyses could unveil molecular mechanisms for phenotypic variability in SCD3. Therefore, nine LFNG missense variants associated with SCD3, c.564C>A, c.583T>C, c.842C>A, c.467T>G, c.856C>T, c.601G>A, c.446C>T, c.521G>A, and c.766G>A, were assessed in vitro for subcellular localization and protein processing. Glycosyltransferase activity was quantified for the first time in the c.583T>C, c.842C>A, and c.446C>T variants. Primarily, our results are the first to satisfy American College of Medical Genetics and Genomics PS3 criteria (functional evidence via well-established assay) for the pathogenicity of c.583T>C, c.842C>A, and c.446C>T, and replicate this evidence for the remaining six variants. Secondly, this work indicates that all variants that prevent Golgi localization also lead to impaired protein processing. It appears that the FRINGE domain is responsible for this phenomenon. Thirdly, our data suggests that variant proximity to the catalytic residue may influence whether LFNG is improperly trafficked and/or enzymatically dysfunctional. Finally, the phenotype of the axial skeleton, but not elsewhere, may be modulated in a variant-specific fashion. More reports are needed to continue testing this hypothesis. We anticipate our data will be used as a basis for discussion of genotype-phenotype correlations in SCD3.

The road to gene manipulation in the mouse: Jean Brachet Memorial Lecture of the International Society of Differentiation (delivered June 21, 2023 at Cold Spring Harbor Laboratory).

Genetic manipulation in mammals has progressed rapidly in the past decade with the advent of CRISPR-Cas gene editing tools, promising profound impacts on the understanding of human development, health and disease. However, many years of research in divergent fields of experimental embryology, genetics, reproduction, molecular biology and transgenic technology laid the groundwork and have played critical roles for this progress. This article details various threads of research and the central role of the laboratory mouse that came together in reaching this point, all from the perspective of a scientist whose research was deeply immersed in the field.

Advances in live imaging early mouse development: exploring the researcher's interdisciplinary toolkit.

Live imaging is an important part of the developmental biologist's armoury of methods. In the case of the mouse embryo, recent advances in several disciplines including embryo culture, microscopy hardware and computational analysis have all contributed to our ability to probe dynamic events during early development. Together, these advances have provided us with a versatile and powerful 'toolkit', enabling us not only to image events during mouse embryogenesis, but also to intervene with them. In this short Spotlight article, we summarise advances and challenges in using live imaging specifically for understanding early mouse embryogenesis.

A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.

An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing ß-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

Fluid-Dynamic Modeling of Flow in Embryonic Tissue Indicates That Lymphatic Valve Location Is Not Consistently Determined by the Local Fluid Shear or Its Gradient.

OBJECTIVE: Intravascular lymphatic valves often occur in proximity to vessel junctions. It is commonly held that disturbed flow at junctions is responsible for accumulation of valve-forming cells (VFCs) at these locations as the initial step in valve creation, and the one which explains the association with these sites. However, evidence in favor is largely limited to cell culture experiments. METHODS: We acquired images of embryonic lymphatic vascular networks from day E16.5, when VFC accumulation has started but the developing valve has not yet altered the local vessel geometry, stained for Prox1, which co-localizes with Foxc2. Using finite-element computational fluid mechanics, we simulated the flow through the networks, under conditions appropriate to this early development stage. Then we correlated the Prox1 distributions with the distributions of simulated fluid shear and shear stress gradient. RESULTS: Across a total of 16 image sets, no consistent correlation was found between Prox1 distribution and the local magnitude of fluid shear, or its positive or negative gradient. CONCLUSIONS: This, the first direct semi-empirical test of the localization hypothesis to interrogate the tissue from in vivo at the critical moment of development, does not support the idea that a feature of the local flow determines valve localization.

Seeking arrangements: cell contact as a cleavage-stage biomarker.

RESEARCH QUESTION: What can three-dimensional cell contact networks tell us about the developmental potential of cleavage-stage human embryos? DESIGN: This pilot study was a retrospective analysis of two Embryoscope imaging datasets from two clinics. An artificial intelligence system was used to reconstruct the three-dimensional structure of embryos from 11-plane focal stacks. Networks of cell contacts were extracted from the resulting embryo three-dimensional models and each embryo's mean contacts per cell was computed. Unpaired t-tests and receiver operating characteristic curve analysis were used to statistically analyse mean cell contact outcomes. Cell contact networks from different embryos were compared with identical embryos with similar cell arrangements. RESULTS: At t4, a higher mean number of contacts per cell was associated with greater rates of blastulation and blastocyst quality. No associations were found with biochemical pregnancy, live birth, miscarriage or ploidy. At t8, a higher mean number of contacts was associated with increased blastocyst quality, biochemical pregnancy and live birth. No associations were found with miscarriage or aneuploidy. Mean contacts at t4 weakly correlated with those at t8. Four-cell embryos fell into nine distinct cell arrangements; the five most common accounted for 97% of embryos. Eight-cell embryos, however, displayed a greater degree of variation with 59 distinct cell arrangements. CONCLUSIONS: Evidence is provided for the clinical relevance of cleavage-stage cell arrangement in the human preimplantation embryo beyond the four-cell stage, which may improve selection techniques for day-3 transfers. This pilot study provides a strong case for further investigation into spatial biomarkers and three-dimensional morphokinetics.

Absence of Ductus Venosus: A Comparison of 2 Distinctive Fetal Autopsy Cases and Embryologic Perspectives.

In fetal circulation, oxygenated blood from the placenta flows through the umbilical vein into the ductus venosus (DV), then enters the inferior vena cava, and subsequently reaches the right atrium of the heart. The DV serves as a shunt, allowing this oxygen-rich blood to bypass the liver. The absence of the DV (ADV), also known as agenesis of the DV, is a rare congenital anomaly. Without a DV, blood from the umbilical vein must follow alternative routes to the heart. In ADV cases, blood from the umbilical vein must follow 1 of 2 primary drainage patterns: either an extrahepatic shunt or an intrahepatic shunt. This report details the antenatal ultrasound and postmortem findings of 2 fetuses diagnosed with ADV by prenatal imaging studies. The first case involved a fetus with a persistent right umbilical vein connected directly to the suprahepatic IVC, accompanied by early obliteration of the left umbilical vein and true agenesis of the DV. This fetus also had additional congenital anomalies. In contrast, the second case involved a fetus with a normal left umbilical vein that entered the liver. However, despite an ultrasound diagnosis of "absence" of the DV, a DV was present, though markedly hypoplastic and probably minimally functional or non-functional. In this case, blood from the umbilical vein likely followed an alternate intrahepatic route through the portal and hepatic veins, before reaching the heart (intrahepatic shunt). These contrasting cases emphasize the heterogeneity of vascular anomalies and embryologic origins captured by the term "ADV." Additionally, the terminology of "absence" or "agenesis" may be misleading in some purported ADV cases. Specifically, in the second case, the DV was not absent; it was markedly hypoplastic instead. This also appears to be the first reported case of a hypoplastic DV in a fetus. Both cases underscore the importance of effective collaboration and clear communication between maternal-fetal medicine specialists and pathologists.

Soranus of Ephesus (98-138 ad) and his contributions on human embryology, paediatrics and obstetric knowledge.

This study describes the life and works of Soranus of Ephesus (98-138 AD) who was born in Ephesus in Asia Minor and he practiced medicine in Alexandria and Rome in the Roman period (1st/2nd century AD). Although the majority of his writings have been lost, his surviving popular scientific textbook, a four-volume treatise on "Gynecology", describes ancient medical practice regarding human embryology, paediatrics and obstetrics.

Development of the human fetal testis: Morphology and expression of cellular differentiation markers.

A comprehensive immunohistochemical ontogeny of the developing human fetal testis has remained incomplete in the literature to date. We collected human fetal testes from 8 to 21 weeks of fetal age, as well as postnatal human testes at minipuberty, pre-pubertal, and pubertal stages. Immunohistochemistry was performed with a comprehensive panel of antigens targeting gonadocytes, Sertoli cells, fetal Leydig cells, peritubular myoid cells, and other hormonal and developmental targets. Testicular cords, precursor structures to seminiferous tubules, developed from 8 to 14 weeks of fetal age, separating the testis into the interstitial and intracordal compartments. Fetal gonadocytes were localized within the testicular cords and evaluated for Testis-Specific Protein Y, Octamer-binding transcription factor 4, Sal-like protein 4, and placental alkaline phosphatase expression. Fetal Sertoli cells were also localized in the testicular cords and evaluated for SRY-box Transcription Factor 9, inhibin, and anti-Mullerian hormone expression. Fetal Leydig cells were present in the interstitium and stained for cytochrome p450c17 and calretinin, while interstitial peritubular myoid cells were examined using smooth muscle α-actin staining. Androgen receptor expression was localized close to the testicular medulla at 8 weeks and then around the testicular cords in the interstitium as they matured in structure. Postnatal staining showed that Testis-Specific Protein Y remained positive of male gonadocytes throughout adulthood. Anti-Mullerian hormone, SRY-box Transcription Factor 9, and Steroidogenic factor 1 are expressed by the postnatal Sertoli cells at all ages examined. Leydig cell markers cytochrome p450c17 and calretinin are expressed during mini-puberty and puberty, but not expressed during the pre-pubertal period. Smooth muscle α-actin and androgen receptor were not expressed during mini-puberty or pre-puberty, but again expressed during the pubertal period. The ontogenic map of the human fetal and postnatal testicular structure and expression patterns described here will serve as a reference for future investigations into normal and abnormal testicular development.

Development of the human ovary: Fetal through pubertal ovarian morphology, folliculogenesis and expression of cellular differentiation markers.

A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation.

High resolution dynamic ultrasound atlas of embryonic and fetal development of the common marmoset.

PURPOSE: The common marmoset (Callithrix jacchus) provides an ideal model to study early development of primates, and an in vivo platform to validate conclusions from in vitro studies of human embryos and embryo models. Currently, however, no established staging atlas of marmoset embryonic development exists. Using high-resolution, longitudinal ultrasound scans on live pregnant marmosets, we present the first dynamic in vivo imaging of entire primate gestation beginning with attachment until the last day before birth. METHODS: Our study unveils the first dynamic images of an in vivo attached mammalian embryo developing in utero, and the intricacies of the delayed development period unique to the common marmoset amongst primates, revealing a window for somatic interventions. RESULTS: Established obstetric and embryologic measurements for each scan were used comparatively with the standardized Carnegie staging of human development to highlight similarities and differences. Our study also allows for tracking the development of major organs. We focus on the ontogeny of the primate heart and brain. Finally, input ultrasound images were used to train deep neural networks to accurately determine the gestational age. All our ultrasounds and staging data recording are posted online so that the atlas can be used as a community resource toward monitoring and managing marmoset breeding colonies. CONCLUSION: The temporal and spatial resolution of ultrasound achieved in this study demonstrates the promise of noninvasive imaging in the marmoset for the in vivo study of primate-specific aspects of embryonic and fetal development.

Flipped online teaching of histology and embryology with design thinking: design, practice and reflection.

BACKGROUND: Flexible hybrid teaching has become the new normal of basic medical education in the postepidemic era. Identifying ways to improve the quality of curriculum teaching and achieve high-level talent training is a complex problem that urgently needs to be solved. Over the course of the past several semesters, the research team has integrated design thinking (DT) into undergraduate teaching to identify, redesign and solve complex problems in achieving curriculum teaching and professional talent training objectives. METHODS: This study is an observational research. A total of 156 undergraduate stomatology students from Jining Medical University in 2021 were selected to participate in two rounds of online flipped teaching using the design thinking EDIPT (empathy, definition, idea, prototype, and test) method. This approach was applied specifically to the chapters on the respiratory system and female reproductive system. Data collection included student questionnaires, teacher-student interviews, and exam scores. GraphPad Prism software was used for data analysis, and the statistical method was conducted by multiple or unpaired t test. RESULTS: According to the questionnaire results, the flipped classroom teaching design developed using design thinking methods received strong support from the majority of students, with nearly 80% of students providing feedback that they developed multiple abilities during the study process. The interview results indicated that teachers generally believed that using design thinking methods to understand students' real needs, define teaching problems, and devise instructional design solutions, along with testing and promptly adjusting the effectiveness through teaching practices, played a highly positive role in improving teaching and student learning outcomes. A comparison of exam scores showed a significant improvement in the exam scores of the class of 2021 stomatology students in the flipped teaching chapters compared to the class of 2020 stomatology students, and this difference was statistically significant. However, due to the limitation of the experimental chapter scope, there was no significant difference in the overall course grades. CONCLUSION: The study explores the application of design thinking in histology and embryology teaching, revealing its positive impact on innovative teaching strategies and students' learning experience in medical education. Online flipped teaching, developed through design thinking, proves to be an effective and flexible method that enhances student engagement and fosters autonomous learning abilities.

Embryological basis of polyorchidism including classification, reproductive potential, and risk of malignancy: A review.

Polyorchidism, a congenital malformation characterized by supernumerary testes (SNTs), is usually revealed incidentally during ultrasound or open scrotal surgery. In the approximately 200 cases so far published in the literature, the left side is affected more often than the right. Despite the rarity of this anomaly, a surgeon must have basic knowledge of its embryological basis and classifications to implement proper treatment and avoid overlooking it, since the consequences could harm the patient. This review summarizes previous classifications. It can be assumed that determining the risk of malignancy, and the level of reproductive potential based on location, vascularization, ductus deferens drainage, and environmental factors (e.g., temperature) affecting the SNTs, indicates the best approach to management. Therefore, we have created a new classification based on previous ones, addressing the aforementioned issues, which will guide the clinician to select the most appropriate treatment.

Introducing AnatomyGPT: A customized artificial intelligence application for anatomical sciences education.

Artificial intelligence (AI) technologies are poised to become an increasingly important part of education in the anatomical sciences. OpenAI has also introduced generative pretrained transformers (GPTs), which are customizable versions of the standard ChatGPT application. There is little research that has explored the potential of GPTs to serve as intelligent tutoring systems for learning the anatomical sciences. The objective of this study was to describe the design and explore the performance of AnatomyGPT, a customized artificial intelligence application intended for anatomical sciences education. The AnatomyGPT application was configured with GPT Builder by uploading open-source textbooks as knowledge sources and by providing pedagogical instructions for how to interact with users. The performance of AnatomyGPT was compared with ChatGPT by evaluating the responses of both applications to prompts of the National Board of Medical Examiners (NBME) sample items with respect to accuracy, rationales, and citations. AnatomyGPT achieved high scores on the NBME sample items for Gross Anatomy, Embryology, Histology, and Neuroscience and scored comparably to ChatGPT. In addition, AnatomyGPT provided several citations in the responses that it generated, while ChatGPT provided none. Both GPTs provided rationales for all sample items. The customized AnatomyGPT application demonstrated preliminary potential as an intelligent tutoring system by generating responses with increased citations as compared with the standard ChatGPT application. The findings of this study suggest that instructors and students may wish to create their own custom GPTs for teaching and learning anatomy. Future research is needed to further develop and characterize the potential of GPTs for anatomy education.

Human Malformed Perinatal Anthropological Crania Contribute to New Insight in the Extension of Bone Malformations in Cranial Development.

INTRODUCTION: We describe five abnormal crania which may provide more diagnostic data for assessment of abnormal crania in newborns. METHODS: Five malformed perinatal crania from the Saxtorphian Collection are described using published prenatal abnormal cranial development criteria. These malformations were compared to normal cranial development arising from the migration of neural crest cells. Visual and photographic investigations were performed. RESULTS: The malformed crania were occipital encephalocele, holoprosencephaly, anencephaly, and two without a recognizable diagnosis. The anthropological crania were malformed in the same regions as formerly observed in fetal pathology. These regions were comparable to fields formed during normal cell migration from the neural crest. This has seemingly not previously been demonstrated. One undiagnosed cranium may represent a Treacher Collins syndrome (Case 3). The other undiagnosed cranium (Case 4) could be from a scaphocephalic specimen. DISCUSSION: Sharp borderlines between malformed and non-malformed regions in cranial syndromes may enable improvement in diagnostics.

External Disruption of Ocular Development in Utero.

The intricate steps of human ocular embryology are impacted by cellular and genetic signaling pathways and a myriad of external elements that can affect pregnancy, such as environmental, metabolic, hormonal factors, medications, and intrauterine infections. This review focuses on presenting some of these factors to recognize the multifactorial nature of ocular development and highlight their clinical significance. This review is based on English-language articles sourced from PubMed, Web of Science, and Google Scholar; keywords searched included "ocular development in pregnancy," "ocular embryology," "maternal nutrition," "ophthalmic change," and "visual system development." While some animal models show the disruption of ocular embryology from these external factors, there are limited post-birth assessments in human studies. Much remains unknown about the precise mechanisms of how these external factors can disrupt normal ocular development in utero, and more significant research is needed to understand the pathophysiology of these disruptive effects further. Findings in this review emphasize the importance of additional research in understanding the dynamic association between factors impacting gestation and neonatal ocular development, particularly in the setting of limited resources.