Developmental Landmarks Research Articles

Using Zebrafish to Screen Developmental Toxicity of Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are found in many consumer and industrial products. While some PFAS, notably perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are developmentally toxic in mammals, the vast majority of PFAS have not been evaluated for developmental toxicity potential. A concentration-response study of 182 unique PFAS chemicals using the zebrafish medium-throughput, developmental vertebrate toxicity assay was conducted to investigate chemical structural identifiers for toxicity. Embryos were exposed to each PFAS compound (≤100 μM) beginning on the day of fertilization. At 6 days post-fertilization (dpf), two independent observers graded developmental landmarks for each larva (e.g., mortality, hatching, swim bladder inflation, edema, abnormal spine/tail, or craniofacial structure). Thirty percent of the PFAS were developmentally toxic, but there was no enrichment of any OECD structural category. PFOS was developmentally toxic (benchmark concentration [BMC] = 7.48 μM); however, other chemicals were more potent: perfluorooctanesulfonamide (PFOSA), N-methylperfluorooctane sulfonamide (N-MeFOSA), ((perfluorooctyl)ethyl)phosphonic acid, perfluoro-3,6,9-trioxatridecanoic acid, and perfluorohexane sulfonamide. The developmental toxicity profile for these more potent PFAS is largely unexplored in mammals and other species. Based on these zebrafish developmental toxicity results, additional screening may be warranted to understand the toxicity profile of these chemicals in other species.

A developmental atlas of male terminalia across twelve species of Drosophila.

How complex morphologies evolve is one of the central questions in evolutionary biology. Observing the morphogenetic events that occur during development provides a unique perspective on the origins and diversification of morphological novelty. One can trace the tissue of origin, emergence, and even regression of structures to resolve murky homology relationships between species. Here, we trace the developmental events that shape some of the most diverse organs in the animal kingdom-the male terminalia (genitalia and analia) of Drosophilids. Male genitalia are known for their rapid evolution with closely related species of the Drosophila genus demonstrating vast variation in their reproductive morphology. We used confocal microscopy to monitor terminalia development during metamorphosis in twelve related species of Drosophila. From this comprehensive dataset, we propose a new staging scheme for pupal terminalia development based on shared developmental landmarks, which allows one to align developmental time points between species. We were able to trace the origin of different substructures, find new morphologies and suggest possible homology of certain substructures. Additionally, we demonstrate that posterior lobe is likely originated prior to the split between the Drosophila melanogaster and the Drosophila yakuba clade. Our dataset opens up many new directions of research and provides an entry point for future studies of the Drosophila male terminalia evolution and development.

Phenotypic Analysis: Assessing Timing of Recessive Prenatal Lethality in Mice.

Once a recessive mutation has been established in a mouse strain in the heterozygous state, the task of phenotypic analysis of the homozygous mutants can begin. This overview leads you through a sequence of steps to determine whether the homozygous mutants are present at birth or whether the mutation causes prenatal lethality. In the case of a prenatal lethality, the time of death of the mutants, which could occur at any time during pre- or postimplanation development, must be firmly established before further phenotypic analysis. Here, we present a detailed plan to efficiently determine the time of prenatal death of the mutants and provide a guide for developmental landmarks to establish how far they progress during gestation. To determine whether or not homozygous mutants are present or normal at any given time point, it is important to recover a sufficient number of embryos. Examples of a simple Chi square test for Mendelian segregation is provided to establish statistical significance for the genotype/phenotype distribution.

Therapeutic Hypothermia Is Limited in Preventing Developmental Impairments after Neonatal Hypoxia-Ischemia

The only current treatment for neonatal hypoxia-ischemia (HI) is therapeutic hypothermia (TH), which still shows some limitations. Specific effects of TH in the several processes involved in brain injury progression remain unclear. In this study, the effects of TH treatment on developmental parameters, behavioral outcomes, and peripheral leukocytes were evaluated in neonatal male and female rats. In P7, animals were submitted to right common carotid artery occlusion followed by hypoxia (8% oxygen). TH was performed by reducing the animal scalp temperature to 32°C for 5 h. Behavioral parameters and developmental landmarks were evaluated. Animals were euthanized at P9 or P21, and cerebral hemispheres, spleen, and thymus were weighed. White blood cells (WBCs) were counted in blood smears. There was a reduction in the weight of the brain hemisphere ipsilateral to the carotid occlusion in HI and TH groups, as well as a reduction in body weight gain and a delay in the opening of the ipsilateral eye. Latency in negative geotaxis was increased by HI at P12. TH did not prevent brain weight loss, developmental impairments, or WBC number changes but prevented negative geotaxis impairment and spleen weight reduction. These data reinforce that a better understanding of the events that occur after HI and TH in both males and females is necessary and would allow the development of more adequate and sex-specific therapeutic approaches.

The hologenome of Daphnia magna reveals possible DNA methylation and microbiome-mediated evolution of the host genome.

Properties that make organisms ideal laboratory models in developmental and medical research are often the ones that also make them less representative of wild relatives. The waterflea Daphnia magna is an exception, by both sharing many properties with established laboratory models and being a keystone species, a sentinel species for assessing water quality, an indicator of environmental change and an established ecotoxicology model. Yet, Daphnia's full potential has not been fully exploited because of the challenges associated with assembling and annotating its gene-rich genome. Here, we present the first hologenome of Daphnia magna, consisting of a chromosomal-level assembly of the D. magna genome and the draft assembly of its metagenome. By sequencing and mapping transcriptomes from exposures to environmental conditions and from developmental morphological landmarks, we expand the previously annotates gene set for this species. We also provide evidence for the potential role of gene-body DNA-methylation as a mutagen mediating genome evolution. For the first time, our study shows that the gut microbes provide resistance to commonly used antibiotics and virulence factors, potentially mediating Daphnia's environmental-driven rapid evolution. Key findings in this study improve our understanding of the contribution of DNA methylation and gut microbiota to genome evolution in response to rapidly changing environments.

Road to sexual maturity: Behavioral event schedule from eclosion to first mating in each sex of Drosophila melanogaster

Animals achieve their first mating through the process of sexual maturation. This study examined the precise and detailed timing of a series of behavioral events, including wing expansion, first feeding, first excretion, and courtship, during sexual maturation from eclosion to first mating in D.melanogaster. We found that the time of first mating is genetically invariant and is not affected by light/dark cycle or food intake after eclosion. We also found sexual dimorphism in locomotor activity after eclosion, with females increasing locomotor activity earlier than males. In addition, we found a time rapidly changing from extremely low to high sexual activity in males post eclosion (named "drastic male courtship arousal" or DMCA). These behavioral traits leading up to the first mating could serve as clear indicators of sexual maturation and establish precisely timed developmental landmarks to explore further the mechanisms underlying the integration of behavioral and physiological sexual maturation.

A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Deep Low-Shot Learning for Biological Image Classification and Visualization From Limited Training Samples.

Predictive modeling is useful but very challenging in biological image analysis due to the high cost of obtaining and labeling training data. For example, in the study of gene interaction and regulation in Drosophila embryogenesis, the analysis is most biologically meaningful when in situ hybridization (ISH) gene expression pattern images from the same developmental stage are compared. However, labeling training data with precise stages is very time-consuming even for developmental biologists. Thus, a critical challenge is how to build accurate computational models for precise developmental stage classification from limited training samples. In addition, identification and visualization of developmental landmarks are required to enable biologists to interpret prediction results and calibrate models. To address these challenges, we propose a deep two-step low-shot learning framework to accurately classify ISH images using limited training images. Specifically, to enable accurate model training on limited training samples, we formulate the task as a deep low-shot learning problem and develop a novel two-step learning approach, including data-level learning and feature-level learning. We use a deep residual network as our base model and achieve improved performance in the precise stage prediction task of ISH images. Furthermore, the deep model can be interpreted by computing saliency maps, which consists of pixel-wise contributions of an image to its prediction result. In our task, saliency maps are used to assist the identification and visualization of developmental landmarks. Our experimental results show that the proposed model can not only make accurate predictions but also yield biologically meaningful interpretations. We anticipate our methods to be easily generalizable to other biological image classification tasks with small training datasets. Our open-source code is available at https://github.com/divelab/lsl-fly.

Stem Cell-Derived Microfluidic Amniotic Sac Embryoid (μPASE).

The microfluidic amniotic sac embryoid (μPASE) is a human pluripotent stem cell (hPSC)-derived multicellular human embryo-like structure with molecular and morphological features resembling the progressive development of the early post-implantation human embryonic sac. The microfluidic device is specifically designed to control the formation of hPSC clusters and expose the clusters to different morphogen environments, allowing the development of μPASEs in a highly controllable, reproducible, and scalable fashion. The μPASE model displays human embryonic developmental landmarks such as lumenogenesis of the epiblast, amniotic cavity formation, and the specification of primordial germ cells and gastrulating cells (or mesendoderm cells). Here, we provide detailed instructions needed to reproduce μPASEs, including the immunofluorescence staining and cell retrieval protocols for characterizing μPASEs obtained under different experimental conditions.

Prenatal zestoretic exposure ameliorates developmental landmarks in rat offspring

Zestoretic, a combination of lisinopril and hydrochlorothiozide used as anti-hypertension drug prescribed even during pregnancy. Role of zestoretic on the developmental landmars of young ones exposed prenatally are yet to be established. Hence, the present investigation initiated to test in Wistar rats. Three groups of inseminated female rats were oral gavaged with zestoretic (lisinopril varied concentrations + hydrochlorothiozide 12.5mg) 25, 50 and 100 mg/Kg body weight (BW) on gestation days 7, 9, 11 and 13, whereas control group with double distilled water. Clinical toxicity found in few dams from 50 and 100 mg/Kg BW received rat shown aggressive behavior during experimentation. The developmental landmarks, such as pinna attachment, ear opening, fur development, eye opening, upper and lower incisor eruption, crown rump length, vaginal opening and testes descend were measured periodically on their postnatal days in all the young ones. The number of live pups in zestoretic 50, 100 mg/Kg BW administered females are significantly less (P

Methimazole-induced gestational hypothyroidism affects the offspring development and differently impairs the conditioned fear in male and female adulthood rodents.

Gestational hypothyroidism is a prevalent disorder in pregnant women and also impairs fetal development with relevant outcomes. One of the outcomes of greatest interest has been rodent fear- and anxiety-like behavior. However, the relationship between maternal hypothyroidism and onset of conditioned fear-related responses in offspring remains controversial. Here, we used a well-validated methimazole-induced gestational hypothyroidism to investigate the behavioral consequences in offspring. Dams were treated with methimazole at 0.02% in drinking water up to gestational Day 9. Maternal body weights and maternal behavior were evaluated, and the puppies ware analyzed for weight gain and physical/behavioral development and assigned for the open field and fear conditioning test. Methimazole-induced gestational hypothyroidism induced loss in maternal and litter weight, increases in maternal behavior, and impairs in offspring developmental landmarks in both male and female rodents. Only male offspring enhanced responsiveness to conditioned fear-like behavior in adulthood.

Transforming growth factor beta (TGFβ) pathway is essential for hypoblast and epiblast development in ovine post-hatching embryos

The developmental failures occurring between blastocyst hatching and implantation in farm ungulates are a major cause of pregnancy losses. At the expanded blastocyst stage, three cell lineages emerge in the embryo: trophoblast, hypoblast and epiblast, the latter being the most vulnerable during post-hatching development. Transforming growth factor beta (TGFβ) signaling pathway is involved in hypoblast and epiblast development; however, previous in vitro functional studies are limited to the expanded blastocyst stage. In this study, we have analyzed the effect of TGFβ inhibition with 10, 20 or 40 μM SB431542 during ovine post-hatching developmental period using a recently developed culture system able to recapitulate major developmental landmarks. We have found a negative effect of TGFβ inhibition on hypoblast and epiblast development that could be partially reverted by Rho-associated protein kinase (ROCK) inhibitor Y-27632. Our findings provide new insights into the molecular networks regulating embryo development beyond the expanded blastocyst and could help to elucidate the causes of early pregnancy losses in farm ungulates.

The temporal dynamics of the sea urchin regulome.

ABSTRACTIn this work, we used Nanostring N-counter technology, to evaluate the mRNA expression level of more than 330 regulatory genes over 34 time points covering the first three days of development of the sea urchin larvae. The hierarchical clustering of the mRNAs expression levels has identified groups corresponding to the major developmental landmarks (e.g. maternal to zygotic transition and gastrulation). Furthermore, comparison with previous experiments indicates high reproducibility of mRNA level temporal dynamics across batches. Finally, we generated an online tool to visualise gene expression during sea urchin larval development. The site can be accessed at and https://www621.lamp.le.ac.uk/nanostring_app/nanostring/.

In vitro culture of ovine embryos up to early gastrulating stages.

ABSTRACTDevelopmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes – trophoblast and hypoblast – and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated ‘The people behind the papers’ interview.

Morphological Variations of Umbilical Cord in Normal Pregnancies among Local Population

Placenta is a unique functional center at the maternal-fetal interface. Aim: To find out the morphological variants of umbilical cord in normal pregnancies was undertaken among local population. Study design: Cross-sectional study. Methodology: Umbilical cords were collected from normal cases of delivery at Services Hospital, Lahore. Simple random sampling (probability) was done to collect hundred placentae. Grossing was done. Tissue samples were taken and processed. Histological observations were undertaken using H&E stained sections. Detailed morphology was studied to find out morphological variant with values ≤ 5%. Data was analyzed using SPSS version 20. Results: Cords that measured < 40cm in length were up to 4%. Cord with no twists was up to 1%, 1% cord with 3.5 twists, 3% cords with 4 twists, and 1% cord with 4.5 twists were present, amount of Wharton's Jelly measured to be 38.46mm2 (1%), 132.66mm2 (2%), 153.85mm2 (2%) and 176.62mm2 (4%), presence of cysts (4%), the vestigial remnants i.e. vitelline and vascular remnants 3% each and squamous metaplasia in 1% of the umbilical cord. Conclusion: Data concluded the presence of morphological variants in the umbilical cord which may reflect the developmental landmarks or other altered phenomenon during the course of pregnancy. Keywords: Umbilical Cord, Vestigial Remnants and Pregnancy.

The Curious Case of Multicellularity in the Volvocine Algae.

The evolution of multicellularity is a major evolutionary transition that underlies the radiation of many species in all domains of life, especially in eukaryotes. The volvocine green algae are an unconventional model system that holds great promise in the field given its genetic tractability, late transition to multicellularity, and phenotypic diversity. Multiple efforts at linking multicellularity-related developmental landmarks to key molecular changes, especially at the genome level, have provided key insights into the molecular innovations or lack thereof that underlie multicellularity. Twelve developmental changes have been proposed to explain the evolution of complex differentiated multicellularity in the volvocine algae. Co-option of key genes, such as cell cycle and developmental regulators has been observed, but with few exceptions, known co-option events do not seem to coincide with most developmental features observed in multicellular volvocines. The apparent lack of “master multicellularity genes” combined with no apparent correlation between gene gains for developmental processes suggest the possibility that many multicellular traits might be the product gene-regulatory and functional innovations; in other words, multicellularity can arise from shared genomic repertoires that undergo regulatory and functional overhauls.

External Scientific Report on the Interpretation of Data from the Developmental Neurotoxicity In Vitro Testing Assays for Use in Integrated Approaches for Testing and Assessment

Developmentally neurotoxic chemicals are a diverse set of substances that have the potential to interfere with the normal development of the nervous system, which, if perturbed without compensation, may lead to adverse effects on the normal development of nervous system structures and/or functions. The current OECD test guideline (TG) 426 requires assessing the impact of fetal and postnatal maternal exposure on the development of: physical and developmental landmarks, clinical observations, and behavioural and neuropathological endpoints (OECD 2007). Use of the in vivo test guideline has been limited due to extensive time, resource costs and animal used. This limited testing, coupled with an increasing need to assess the hazards of hundreds of pesticides and thousands of industrial chemicals, has resulted in calls for the development and use of in vitro methods. International efforts have led to the development of the Developmental Neurotoxicity In Vitro Battery (DNT IVB). The purpose of the present document is to provide guidance on the interpretation of data developed with the DNT IVB for use in regulatory decisions. The major aims are to describe the assays that comprise the battery in terms of neurodevelopment, provide criteria that allows evaluation of the relevance of the data to developmental neurotoxicity, and to assist in the determination of the degree of certainty in any positive or negative findings to better inform use of DNT in vitro data in regulatory hazard determinations.

O-045 Synthetic human embryo-like structures: a new paradigm for human embryology

Abstract text Early human development remains mysterious and very difficult to study. Recent advances in mammalian embryology, stem cell biology, organoid technology, and bioengineering have contributed to a significant interest in bottom-up, synthetic stem cell-derived models of human development (or embryoids). The controllability and reproducibility of human embryoids coupled with the ease of genetically modifying stem cell lines, the ability to manipulate culture conditions and the simplicity of live imaging make them robust and attractive systems to disentangle cellular behaviors and signaling interactions that drive human embryogenesis. In this talk, I will describe our effort in using human pluripotent stem cells (hPSCs) to develop tractable experimental models of the peri-implantation embryonic development and neurulation. The peri-implantation human embryoids developed by us recapitulate key early post-implantation developmental landmarks successively, including pro-amniotic cavity formation, amniotic ectoderm-epiblast patterning, primordial germ cell specification, and development of the primitive streak with controlled anteroposterior polarity. I will further discuss an hPSC-based neuroectoderm patterning model to recapitulate the formation of the neural plate and another patterned neural tube model with fully defined anterior-posterior and dorsal-ventral axes.

Reconstitution of Human Peri-Implantation Embryogenesis With Integrated Stem Cell-Based Embryo Models

Studies of cultured human embryos have provided insights into human peri-implantation embryogenesis but are limited by ethical and technical constraints. Here we assembled a 3D integrated embryo model (IEM) using naive human embryonic stem cells (hESCs) and hESC-derived proliferating trophoblasts. By comparing phenotypic cell fate and comprehensive single-cell transcriptome of IEMs with 3D-cultured human embryos, we reveal that IEMs with embryonic and extra-embryonic cell types mimic developmental landmarks and 3D morphological features of natural human post­­­ ­­-implantation embryos, including pluripotent state transitions, epithelial-mesenchymal transition, anterior-posterior axis formation, amniotic cavity, yolk sac, bilaminar embryonic disc, extra-embryonic mesenchyme, primitive streak and primordial germ cells. Using IEMs, we unravel composition and gene expression patterns of embryonic lineages, and that trophoblasts secret WNT, BMP and TGFβ/Activin signals to instruct post-implantation embryogenesis . Our findings show that IEMs provide a scalable and tractable model for decoding regulatory mechanisms of human peri-implantation embryogenesis.

Embryonic development in the acoel Hofstenia miamia.

Acoels are marine worms that belong to the phylum Xenacoelomorpha, a deep-diverging bilaterian lineage. This makes acoels an attractive system for studying the evolution of major bilaterian traits. Thus far, acoel development has not been described in detail at the morphological and transcriptomic levels in a species in which functional genetic studies are possible. We present a set of developmental landmarks for embryogenesis in the highly regenerative acoel Hofstenia miamia. We generated a developmental staging atlas from zygote to hatched worm based on gross morphology, with accompanying bulk transcriptome data. Hofstenia embryos undergo a stereotyped cleavage program known as duet cleavage, which results in two large vegetal pole 'macromeres' and numerous small animal pole 'micromeres'. These macromeres become internalized as micromere progeny proliferate and move vegetally. We also noted a second, previously undescribed, cell-internalization event at the animal pole, following which we detected major body axes and tissues corresponding to all three germ layers. Our work on Hofstenia embryos provides a resource for mechanistic investigations of acoel development, which will yield insights into the evolution of bilaterian development and regeneration.