Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates.

The early identification of teratogens in humans and animals is mandatory for drug discovery and development. Zebrafish has emerged as an alternative model to traditional preclinical models for predicting teratogenicity and other potential chemical-induced toxicity hazards. To prove its predictivity, we exposed zebrafish embryos from 0 to 96 h post fertilization to a battery of 31 compounds classified as teratogens or non-teratogens in mammals. The teratogenicity score was based on the measurement of 16 phenotypical parameters, namely heart edema, pigmentation, body length, eye size, yolk size, yolk sac edema, otic vesicle defects, otoliths defects, body axis defects, developmental delay, tail bending, scoliosis, lateral fins absence, hatching ratio, lower jaw malformations and tissue necrosis. Among the 31 compounds, 20 were detected as teratogens and 11 as non-teratogens, resulting in 94.44 % sensitivity, 90.91 % specificity and 87.10 % accuracy compared to rodents. These percentages decreased slightly when referred to humans, with 87.50 % sensitivity, 81.82 % specificity and 74.19 % accuracy, but allowed an increase in the prediction levels reported by rodents for the same compounds. Positive compounds showed a high correlation among teratogenic parameters, pointing out at general developmental delay as major cause to explain the physiological/morphological malformations. A more detailed analysis based on deviations from main trends revealed potential specific modes of action for some compounds such as retinoic acid, DEAB, ochratoxin A, haloperidol, warfarin, valproic acid, acetaminophen, dasatinib, imatinib, dexamethasone, 6-aminonicotinamide and bisphenol A. The high degree of predictivity and the possibility of applying mechanistic approaches makes zebrafish a powerful model for screening teratogenicity.

CRISPR Meets Zebrafish: Accelerating the Discovery of New Therapeutic Targets.

Bringing a new drug to the market costs an average of US$2.6 billion and takes more than 10 years from discovery to regulatory approval. Despite the need to reduce cost and time to increase productivity, pharma companies tend to crowd their efforts in the same indications and drug targets. This results in the commercialization of drugs that share the same mechanism of action (MoA) and, in many cases, equivalent efficacies among them-an outcome that helps neither patients nor the balance sheet of the companies trying to bring therapeutics to the same patient population. Indeed, the discovery of new therapeutic targets, based on a deeper understanding of the disease biology, would likely provide more innovative MoAs and potentially greater drug efficacies. It would also bring better chances for identifying appropriate treatments according to the patient's genetic stratification. Nowadays, we count with an enormous amount of unprocessed information on potential disease targets that could be extracted from omics data obtained from patient samples. In addition, hundreds of pharmacological and genetic screenings have been performed to identify innovative drug targets. Traditionally, rodents have been the animal models of choice to perform functional genomic studies. The high experimental cost, combined with the low throughput provided by those models, however, is a bottleneck for discovering and validating novel genetic disease associations. To overcome these limitations, we propose that zebrafish, in conjunction with the use of CRISPR/Cas9 genome-editing tools, could streamline functional genomic processes to bring biologically relevant knowledge on innovative disease targets in a shorter time frame.

ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer.

The xenograft of human cancer cells in model animals is a powerful tool for understanding tumor progression and metastatic potential. Mice represent a validated host, but their use is limited by the elevated experimental costs and low throughput. To overcome these restrictions, zebrafish larvae might represent a valuable alternative. Their small size and transparency allow the tracking of transplanted cells. Therefore, tumor growth and early steps of metastasis, which are difficult to evaluate in mice, can be addressed. In spite of its advantages, the use of this model has been hindered by lack of experimental homogeneity and validation. Considering these facts, the aim of our work was to standardize, automate, and validate a zebrafish larvae xenograft assay with increased translatability and higher drug screening throughput. The ZeOncoTest reliability is based on the optimization of different experimental parameters, such as cell labeling, injection site, automated individual sample image acquisition, and analysis. This workflow implementation finally allows a higher precision and experimental throughput increase, when compared to previous reports. The approach was validated with the breast cancer cell line MDA-MB-231, the colorectal cancer cells HCT116, and the prostate cancer cells PC3; and known drugs, respectively RKI-1447, Docetaxel, and Mitoxantrone. The results recapitulate growth and invasion for all tested tumor cells, along with expected efficacy of the compounds. Finally, the methodology has proven useful for understanding specific drugs mode of action. The insights gained bring a step further for zebrafish larvae xenografts to enter the regulated preclinical drug discovery path.

Yap/Taz-TEAD activity links mechanical cues to progenitor cell behavior during zebrafish hindbrain segmentation.

Cells perceive their microenvironment through chemical and physical cues. However, how the mechanical signals are interpreted during embryonic tissue deformation to result in specific cell behaviors is largely unknown. The Yap/Taz family of transcriptional co-activators has emerged as an important regulator of tissue growth and regeneration, responding to physical cues from the extracellular matrix, and to cell shape and actomyosin cytoskeletal changes. In this study, we demonstrate the role of Yap/Taz-TEAD activity as a sensor of mechanical signals in the regulation of the progenitor behavior of boundary cells during zebrafish hindbrain compartmentalization. Monitoring of in vivo Yap/Taz activity during hindbrain segmentation indicated that boundary cells responded to mechanical cues in a cell-autonomous manner through Yap/Taz-TEAD activity. Cell-lineage analysis revealed that Yap/Taz-TEAD boundary cells decreased their proliferative activity when Yap/Taz-TEAD activity ceased, which preceded changes in their cell fate from proliferating progenitors to differentiated neurons. Functional experiments demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining progenitor features in the hindbrain boundary cell population.

Comparison of Zebrafish Larvae and hiPSC Cardiomyocytes for Predicting Drug-Induced Cardiotoxicity in Humans.

Cardiovascular drug toxicity is responsible for 17% of drug withdrawals in clinical phases, half of post-marketed drug withdrawals and remains an important adverse effect of several marketed drugs. Early assessment of drug-induced cardiovascular toxicity is mandatory and typically done in cellular systems and mammals. Current in vitro screening methods allow high-throughput but are biologically reductionist. The use of mammal models, which allow a better translatability for predicting clinical outputs, is low-throughput, highly expensive, and ethically controversial. Given the analogies between the human and the zebrafish cardiovascular systems, we propose the use of zebrafish larvae during early drug discovery phases as a balanced model between biological translatability and screening throughput for addressing potential liabilities. To this end, we have developed a high-throughput screening platform that enables fully automatized in vivo image acquisition and analysis to extract a plethora of relevant cardiovascular parameters: heart rate, arrhythmia, AV blockage, ejection fraction, and blood flow, among others. We have used this platform to address the predictive power of zebrafish larvae for detecting potential cardiovascular liabilities in humans. We tested a chemical library of 92 compounds with known clinical cardiotoxicity profiles. The cross-comparison with clinical data and data acquired from human induced pluripotent stem cell cardiomyocytes calcium imaging showed that zebrafish larvae allow a more reliable prediction of cardiotoxicity than cellular systems. Interestingly, our analysis with zebrafish yields similar predictive performance as previous validation meta-studies performed with dogs, the standard regulatory preclinical model for predicting cardiotoxic liabilities prior to clinical phases.

Multiplex Analysis Platform for Endocrine Disruption Prediction Using Zebrafish.

Small fish are an excellent experimental model to screen endocrine-disrupting compounds, but current fish-based assays to detect endocrine disruption have not been standardized yet, meaning that there is not consensus on endpoints and biomarkers to be measured. Moreover, exposure conditions may vary depending on the species used as the experimental model and the endocrine pathway evaluated. At present, a battery of a wide range of assays is usually needed for the complete assessment of endocrine activities. With the aim of providing a simple, robust, and fast assay to assess endocrine-disrupting potencies for the three major endocrine axes, i.e., estrogens, androgens, and thyroid, we propose the use of a panel of eight gene expression biomarkers in zebrafish larvae. This includes brain aromatase (cyp19a1b) and vitellogenin 1 (vtg1) for estrogens, cytosolic sulfotransferase 2 family 2 (sult2st3) and cytochrome P450 2k22 (cyp2k22) for androgens, and thyroid peroxidase (tpo), transthyretin (ttr), thyroid receptor α (trα), and iodothyronine deiodinase 2 (dio2) for thyroid metabolism. All of them were selected according to their responses after exposure to the natural ligands 17ß-estradiol, testosterone, and 3,3',5-triiodo-L-thyronine (T3), respectively, and subsequently validated using compounds reported as endocrine disruptors in previous studies. Cross-talk effects were also evaluated for all compounds.

Loss of the sphingolipid desaturase DEGS1 causes hypomyelinating leukodystrophy.

Sphingolipid imbalance is the culprit in a variety of neurological diseases, some affecting the myelin sheath. We have used whole-exome sequencing in patients with undetermined leukoencephalopathies to uncover the endoplasmic reticulum lipid desaturase DEGS1 as the causative gene in 19 patients from 13 unrelated families. Shared features among the cases include severe motor arrest, early nystagmus, dystonia, spasticity, and profound failure to thrive. MRI showed hypomyelination, thinning of the corpus callosum, and progressive thalamic and cerebellar atrophy, suggesting a critical role of DEGS1 in myelin development and maintenance. This enzyme converts dihydroceramide (DhCer) into ceramide (Cer) in the final step of the de novo biosynthesis pathway. We detected a marked increase of the substrate DhCer and DhCer/Cer ratios in patients' fibroblasts and muscle. Further, we used a knockdown approach for disease modeling in Danio rerio, followed by a preclinical test with the first-line treatment for multiple sclerosis, fingolimod (FTY720, Gilenya). The enzymatic inhibition of Cer synthase by fingolimod, 1 step prior to DEGS1 in the pathway, reduced the critical DhCer/Cer imbalance and the severe locomotor disability, increasing the number of myelinating oligodendrocytes in a zebrafish model. These proof-of-concept results pave the way to clinical translation.

Combining Zebrafish and CRISPR/Cas9: Toward a More Efficient Drug Discovery Pipeline.

The use of zebrafish larvae in basic and applied research has grown exponentially during the last 20 years. The reasons for this success lay in its specific experimental advantages: on the one hand, the small size, the large number of progeny and the fast life cycle greatly facilitate large-scale approaches while maintaining 3Rs amenability; on the other hand, high genetic and physiological homology with humans and ease of genetic manipulation make zebrafish larvae a highly robust model for understanding human disease. Together, these advantages allow using zebrafish larvae for performing high-throughput research, both in terms of chemical and genetic phenotypic screenings. Therefore, the zebrafish larva as an animal model is placed between more reductionist in vitro high-throughput screenings and informative but low-throughput preclinical assays using mammals. However, despite its biological advantages and growing translational validation, zebrafish remains scarcely used in current drug discovery pipelines. In a context in which the pharmaceutical industry is facing a productivity crisis in bringing new drugs to the market, the combined advantages of zebrafish and the CRISPR/Cas9 system, the most powerful technology for genomic editing to date, has the potential to become a valuable tool for streamlining the generation of models mimicking human disease, the validation of novel drug targets and the discovery of new therapeutics. This review will focus on the most recent advances on CRISPR/Cas9 implementation in zebrafish and all their potential uses in biomedical research and drug discovery.

Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation.

Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific cis-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish.

Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening is becoming an important tool to assess the toxicity and efficacy of novel drugs. This animal model has, from early developmental stages, fully functional organs from a physiological point of view. Thus, drug-induced organ-toxicity can be detected in larval stages, allowing a high predictive power on possible human drug-induced liabilities. Hence, zebrafish can bridge the gap between preclinical in vitro safety assays and rodent models in a fast and cost-effective manner. ZeGlobalTox is an innovative assay that sequentially integrates in vivo cardio-, neuro-, and hepatotoxicity assessment in the same animal, thus impacting strongly in the 3Rs principles. It Reduces, by up to a third, the number of animals required to assess toxicity in those organs. It Refines the drug toxicity evaluation through novel physiological parameters. Finally, it might allow the Replacement of classical species, such as rodents and larger mammals, thanks to its high predictivity (Specificity: 89%, Sensitivity: 68% and Accuracy: 78%).

Cell segregation in the vertebrate hindbrain: a matter of boundaries.

Segregating cells into compartments during embryonic development is essential for growth and pattern formation. In the developing hindbrain, boundaries separate molecularly, physically and neuroanatomically distinct segments called rhombomeres. After rhombomeric cells have acquired their identity, interhombomeric boundaries restrict cell intermingling between adjacent rhombomeres and act as signaling centers to pattern the surrounding tissue. Several works have stressed the relevance of Eph/ephrin signaling in rhombomeric cell sorting. Recent data have unveiled the role of this pathway in the assembly of actomyosin cables as an important mechanism for keeping cells from different rhombomeres segregated. In this Review, we will provide a short summary of recent evidences gathered in different systems suggesting that physical actomyosin barriers can be a general mechanism for tissue separation. We will discuss current evidences supporting a model where cell-cell signaling pathways, such as Eph/ephrin, govern compartmental cell sorting through modulation of the actomyosin cytoskeleton and cell adhesive properties to prevent cell intermingling.

Switching to zebrafish neurobehavioral models: The obsessive-compulsive disorder paradigm.

Obsessive-compulsive disorder (OCD) is the tenth most disabling illness of any kind. OCD stands as a paradigm for complex neurobehavioral disorders due to its polygenic origin. It presents heterogenic clinical presentation, variable disease onset, progression and treatment responses, what makes its understanding a major neuropsychiatric challenge. Like with other neurobehavioral disorders, animal models are essential tools for decoding OCD genetic complexity, understanding its biological base and discovering novel treatments and diagnostic methods. 20 years of rodent OCD modeling have helped to understand the disease better, but multiple questions remain regarding OCD. Innovative whole genome sequencing (WGS) approaches might provide important answers on OCD risk associated genes. However, exploiting those large data sets through the use of traditional animal models is costly and time consuming. Zebrafish might be an appropriate animal model to streamline the pipeline of gene functional validation. This animal model shows several advantages versus rodent models, such as faster and cheaper genetic manipulation, strong impact on the 3Rs implementation, behavioral phenotypic reproducibility of OCD-like behaviors (obsessions and compulsions) and feasibility to develop high-throughput assays for novel OCD drug therapies discovery. In conclusion, zebrafish could be an innovative and relevant model for understanding OCD.

Cell segregation in the vertebrate hindbrain relies on actomyosin cables located at the interhombomeric boundaries.

Segregating cells into compartments during embryonic development is essential for growth and pattern formation. Physical mechanisms shaping compartment boundaries were recently explored in Drosophila, where actomyosin-based barriers were revealed to be important for keeping cells apart. In vertebrates, interhombomeric boundaries are straight interfaces, which often serve as signaling centers that pattern the surrounding tissue. Here, we demonstrate that in the hindbrain of zebrafish embryos cell sorting sharpens the molecular boundaries and, once borders are straight, actomyosin barriers are key to keeping rhombomeric cells segregated. Actomyosin cytoskeletal components are enriched at interhombomeric boundaries, forming cable-like structures in the apical side of the neuroepithelial cells by the time morphological boundaries are visible. When myosin II function is inhibited, cable structures do not form, leading to rhombomeric cell mixing. Downregulation of EphA4a compromises actomyosin cables and cells with different rhombomeric identity intermingle, and the phenotype is rescued enhancing myosin II activity. Moreover, enrichment of actomyosin structures is obtained when EphA4 is ectopically expressed in even-numbered rhombomeres. These findings suggest that mechanical barriers act downstream of EphA/ephrin signaling to segregate cells from different rhombomeres.

Distinct regulatory mechanisms act to establish and maintain Pax3 expression in the developing neural tube.

Pattern formation in developing tissues is driven by the interaction of extrinsic signals with intrinsic transcriptional networks that together establish spatially and temporally restricted profiles of gene expression. How this process is orchestrated at the molecular level by genomic cis-regulatory modules is one of the central questions in developmental biology. Here we have addressed this by analysing the regulation of Pax3 expression in the context of the developing spinal cord. Pax3 is induced early during neural development in progenitors of the dorsal spinal cord and is maintained as pattern is subsequently elaborated, resulting in the segregation of the tissue into dorsal and ventral subdivisions. We used a combination of comparative genomics and transgenic assays to define and dissect several functional cis-regulatory modules associated with the Pax3 locus. We provide evidence that the coordinated activity of two modules establishes and refines Pax3 expression during neural tube development. Mutational analyses of the initiating element revealed that in addition to Wnt signaling, Nkx family homeodomain repressors restrict Pax3 transcription to the presumptive dorsal neural tube. Subsequently, a second module mediates direct positive autoregulation and feedback to maintain Pax3 expression. Together, these data indicate a mechanism by which transient external signals are converted into a sustained expression domain by the activities of distinct regulatory elements. This transcriptional logic differs from the cross-repression that is responsible for the spatiotemporal patterns of gene expression in the ventral neural tube, suggesting that a variety of circuits are deployed within the neural tube regulatory network to establish and elaborate pattern formation.

Retinoid acid specifies neuronal identity through graded expression of Ascl1.

Cell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals, two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively. These neurons have different physiological properties, functions, and connectivity. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1, whereas V3 neurons express Sim1. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.

Use of zebrafish embryos for small molecule screening related to cancer.

The introduction of mechanism-based targeted therapies to treat human cancers is fruit of decades of research into the molecular basis of cancer pathogenesis. Despite the growing knowledge about the molecular mechanisms governing its causes and progression, there is a lack of effective treatments for many types of cancer. The expensive and time-consuming preclinical pipeline for testing molecules slows the discovery of new therapies. Therefore, it is important to consider alternative methodologies both for accelerating therapeutic discovery and reducing costs. In that regard, zebrafish is becoming an attractive model for fast and efficient drug screening. Its use has expanded to many disease research areas, and the postgenomic era has led to the progression of functional studies and boosted the development of general databases, such as ZFIN, and the emergence of more specialized ones, including several catalogues of transgenic reporter screens. Taken together, they provide to the scientific community many tools that could be used for drug discovery. The use of zebrafish in cancer drug screenings could help to economize time and resources even more if we rationalize its use: we could use embryonic screens to identify drugs that address general hallmarks of cancer, and use adults for finding molecules that target specific cancer models.

Signalling from hindbrain boundaries regulates neuronal clustering that patterns neurogenesis.

During central nervous system development, neural progenitors are patterned to form discrete neurogenic and non-neurogenic zones. In the zebrafish hindbrain, neurogenesis is organised by Fgf20a emanating from neurons located at each segment centre that inhibits neuronal differentiation in adjacent progenitors. Here, we have identified a molecular mechanism that clusters fgf20a-expressing neurons in segment centres and uncovered a requirement for this positioning in the regulation of neurogenesis. Disruption of hindbrain boundary cell formation alters the organisation of fgf20a-expressing neurons, consistent with a role of chemorepulsion from boundaries. The semaphorins Sema3fb and Sema3gb, which are expressed by boundary cells, and their receptor Nrp2a are required for clustering of fgf20a-expressing neurons at segment centres. The dispersal of fgf20a-expressing neurons that occurs following the disruption of boundaries or of Sema3fb/Sema3gb signalling leads to reduced FGF target gene expression in progenitors and an increased number of differentiating neurons. Sema3 signalling from boundaries thus links hindbrain segmentation to the positioning of fgf20a-expressing neurons that regulates neurogenesis.

Neuronal subtype specification within a lineage by opposing temporal feed-forward loops.

Neural progenitors generate distinct cell types at different stages, but the mechanisms controlling these temporal transitions are poorly understood. In the Drosophila CNS, a cascade of transcription factors, the "temporal gene cascade," has been identified that acts to alter progenitor competence over time. However, many CNS lineages display broad temporal windows, and it is unclear how broad windows progress into subwindows that generate unique cell types. We have addressed this issue in an identifiable Drosophila CNS lineage and find that a broad castor temporal window is subdivided by two different feed-forward loops, both of which are triggered by castor itself. The first loop acts to specify a unique cell fate, whereas the second loop suppresses the first loop, thereby allowing for the generation of alternate cell fates. This mechanism of temporal and "subtemporal" genes acting in opposing feed-forward loops may be used by many stem cell lineages to generate diversity.

Temporal and spatial windows delimit activation of the outer ring of wingless in the Drosophila wing.

Extracellular signalling molecules play many roles in the development of higher organisms. They are used reiteratively in different tissues and stages, but the response of the receiving cells is controlled in a context dependent manner. The pattern of expression of the signalling molecule Wingless/WNT in Drosophila is extraordinarily complex. We have studied the mechanism that controls its expression and function in the outer ring of the Drosophila wing hinge. Our findings indicate that wingless expression is controlled by a dual mechanism: its initial activation requires the product of zinc finger homeodomain 2 and is subsequently repressed by the product of the gene complex elbow/no ocelli. This tight regulation restricts the activation of wingless temporally and spatially. Later in development, wingless expression is maintained by an autoregulatory loop that involves the product of homothorax. We have analyzed the phenotype of a wingless allelic combination that specifically removes the outer ring, and our results show that Wingless is required to promote local proliferation of the wing base cells. Thus, cell proliferation in the proximal-distal axis is controlled by the sequential activation of wingless in the inner ring and the outer ring at different stages of development.