The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Toxicologic Pathology Forum: Opinion on Approaches for Reporting Toxic and Adverse Dose Levels in Nonclinical Toxicology Studies Supporting the Development of Anticancer Pharmaceuticals.

The advancement of an investigational new drug in humans is a significant developmental milestone. In first-in-human (FIH)-enabling toxicology studies, the highest dose without a test article-related adverse effect (no-observed-adverse-effect-level [NOAEL]) serves as the basis for deriving a safe FIH starting dose. For anticancer pharmaceuticals, the FIH dose may be calculated using the highest non-severely toxic dose (HNSTD) in nonrodent models or the dose severely toxic to 10% (STD10) in rodents. Given the practice of reporting the NOAEL, but the lack of regulatory requirements to do so for anticancer pharmaceuticals, we conducted an informal survey of 20 companies to answer the question "How is our industry reporting toxic/adverse dose levels in FIH-enabling toxicology studies for anticancer indications?" The data indicated 4 reporting approaches, each providing a path to regulatory acceptance. Within the integrated toxicology study report, 45% of respondents report the HNSTD/STD10, 25% report the NOAEL, 20% report both the HNSTD/STD10 and NOAEL, and 10% do not define either, reserving definitions for regulatory submissions. One reporting approach may be preferred over another for reasons including consistency across indications, repurposing pharmaceuticals, regulatory feedback, or simplicity. The reporting approach should be defined in advance of study initiation, and the pathologist should provide context to support the chosen approach.

Spontaneous Findings in the Reproductive System of Sexually Mature Male Cynomolgus Macaques.

Sexually mature nonhuman primates are often used in nonclinical safety testing when evaluating biopharmaceuticals; however, there is limited information in historical control databases or in the published literature on the spontaneous findings in the male reproductive system. This review evaluated digital slides from the male reproductive tract (testes, epididymides, prostate, and seminal vesicles) in sexually mature cynomolgus macaques (Macaca fascicularis; n = 255) from vehicle control groups in nonclinical toxicology studies and compared the observations with body weight, organ weight, and geographical origin. The most common microscopic findings were hypospermatogenesis and tubular dilatation in the testes; inflammatory cell infiltrate, cellular debris, and decreased sperm in the epididymides; inflammatory cell infiltrate and acinar dilatation in the prostate; and corpora amylacea and atrophy in the seminal vesicles. There were a few correlative observations in animals when grouped by weight or geographical origin: animals with lower terminal body weights (<5 kg) often displayed features of late puberty despite having sperm in the epididymis, while animals originating from Mauritius had a lower incidence of inflammatory cell infiltrates than those from Southeast Asia/China. This review provides incidence, descriptions, and photomicrographs of the common spontaneous microscopic findings in the reproductive system of mature male cynomolgus macaques.

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat.

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

Publication Categories in Toxicologic Pathology.

Toxicologic Pathology is the official journal of the Society of Toxicologic Pathology (STP), the British Society of Toxicological Pathology, and the European STP (ESTP). Toxicologic Pathology publishes articles related to topics in various aspects of toxicologic pathology such as anatomic pathology, clinical pathology, experimental pathology, and biomarker research. Publications include society-endorsed Best Practice/Position and Points to Consider publications and ESTP Expert Workshop articles that are relevant to toxicologic pathology and scientific regulatory processes, Opinion articles under the banner of the STP Toxicologic Pathology Forum, Original Articles, Review Articles (unsolicited/contributed, mini, and invited), Brief Communications, Letters to the Editor, Meeting Reports, and Book Reviews. This article provides details on the various publication categories in Toxicologic Pathology and will serve as a reference for authors and readers.

Scientific and Regulatory Policy Committee Best Practices: Documentation of Sexual Maturity by Microscopic Evaluation in Nonclinical Safety Studies.

The sexual maturity status of animals in nonclinical safety studies can have a significant impact on the microscopic assessment of the reproductive system, the interpretation of potential test article-related findings, and ultimately the assessment of potential risk to humans. However, the assessment and documentation of sexual maturity for animals in nonclinical safety studies is not conducted in a consistent manner across the pharmaceutical and chemical industries. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology convened an international working group of pathologists and nonclinical safety scientists with expertise in the reproductive system, pathology nomenclature, and Standard for Exchange of Nonclinical Data requirements. This article describes the best practices for documentation of the light microscopic assessment of sexual maturity in males and females for both rodent and nonrodent nonclinical safety studies. In addition, a review of the microscopic features of the immature, peripubertal, and mature male and female reproductive system and general considerations for study types and reporting are provided to aid the study pathologist tasked with documentation of sexual maturity.

Phosphatidylinositol 3-Kinase δ-Specific Inhibitor-Induced Changes in the Ovary and Testis in the Sprague Dawley Rat and Cynomolgus Monkey.

Phosphatidylinositol 3-kinase (PI3K) δ is a lipid kinase primarily found in leukocytes, which regulates important cell functions. AMG2519493 was a PI3K δ-specific inhibitor in development for treatment of various inflammatory diseases. AMG2519493-related changes in the male and/or female reproductive organs were observed in the 1- and 3-month oral repeat dose toxicology studies in the rat and cynomolgus monkey. Hemorrhagic corpora lutea cysts and increased incidence of corpora lutea cysts without hemorrhage were observed in the ovaries at supra pharmacological doses in the rat. A decrease in seminiferous germ cells in the testis, indicative of spermatogenesis maturation arrest, was observed in both the rat and cynomolgus monkey. Although the characteristics were comparable, the drug systemic exposures associated with the testicular changes were very different between the 2 species. In the rat, the testicular change was only observed at supra pharmacologic exposure. Isotype assessment of PI3K signaling in rat spermatogonia in vitro indicated a role for PI3K ß, but not δ, in the c Kit/PI3K/protein kinase B signaling pathway. Therefore, changes in both the ovary and testis of the rat were considered due to off target effect as they only occurred at suprapharmacologic exposure. In contrast, the testicular changes in the cynomolgus monkey (decrease in seminiferous germ cells) occurred at very low doses associated with PI3K δ-specific inhibition, indicating that the PI3K δ isoform may be important in spermatogenesis maturation in the cynomolgus monkey. Our results suggest species-related differences in PI3K isoform-specific control on reproductive organs.

Nonclinical Safety Profile of Sotorasib, a KRASG12C-Specific Covalent Inhibitor for the Treatment of KRAS p.G12C-Mutated Cancer.

Sotorasib is a first-in-class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. A comprehensive nonclinical safety assessment package, including secondary/safety pharmacology and toxicology studies, was conducted to support the marketing application for sotorasib. Sotorasib was negative in a battery of genotoxicity assays and negative in an in vitro phototoxicity assay. Based on in vitro assays, sotorasib had no off-target effects against various receptors, enzymes (including numerous kinases), ion channels, or transporters. Consistent with the tumor-specific target distribution (ie, KRASG12C), there were no primary pharmacology-related on-target effects identified. The kidney was identified as a target organ in the rat but not the dog. Renal toxicity in the rat was characterized by tubular degeneration and necrosis restricted to a specific region suggesting that the toxicity was attributed to the local formation of a putative toxic reactive metabolite. In the 3-month dog study, adaptive changes of hepatocellular hypertrophy due to drug metabolizing enzyme induction were observed in the liver that was associated with secondary effects in the pituitary and thyroid gland. Sotorasib was not teratogenic and had no direct effect on embryo-fetal development in the rat or rabbit. Human, dog, and rat circulating metabolites, M24, M10, and M18, raised no clinically relevant safety concerns based on the general toxicology studies, primary/secondary pharmacology screening, an in vitro human ether-à-go-go-related gene assay, or mutagenicity assessment. Overall, the results of the nonclinical safety program support a high benefit/risk ratio of sotorasib for the treatment of patients with KRAS p.G12C-mutated tumors.

Phosphatidylinositol 3-Kinase δ Inhibitor-Induced Immunomodulation and Secondary Opportunistic Infection in the Cynomolgus Monkey (Macaca fascicularis).

Phosphatidylinositol 3-kinases (PI3Ks) regulate intracellular signaling events for multiple cell surface receptors. Phosphatidylinositol 3-kinase δ, 1 of 4 class I PI3K isoforms, is primarily found in leukocytes and regulates immune cell functions. Here, we report changes in the immune and digestive systems that were associated with AMG2519493, a highly selective small-molecule PI3Kδ inhibitor. Following 1- or 3-month oral repeat dosing in the cynomolgus monkey, changes were observed in circulating B cells, lymphoid tissues (spleen, lymph nodes, gut-associated lymphoid tissue, tonsil), and the digestive tract. Decreased circulating B cells and lymphoid cellularity in B cell-rich zones in lymphoid tissues were attributed to the intended pharmacologic activity of AMG2519493. Dose- and duration-dependent digestive system toxicity was characterized by inflammation in the large intestine and secondary opportunistic infections restricted to the digestive tract. Digestive tract changes were associated with moribundity and mortality at high-dose levels, and the effect level decreased with increased duration of exposure. These observations demonstrate the role of PI3Kδ in regulation of the immune system and of host resistance to opportunistic infections of the digestive tract.

Variability of Spleen and Mesenteric Lymph Node in Control Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Safety Studies: A Retrospective Assessment.

We assessed the variability of spleen and mesenteric lymph node (MLN) microscopic observations and the correlations of these observations with other study data from 478 control cynomolgus monkeys from 53 routine nonclinical safety studies. Spleen weight parameters (absolute and relative to body or brain weights) were highly variable both within a control group on an individual study (up to 5.11-fold) and among animals with the same light microscopic observation. Grades for microscopic observations were also highly variable. The most frequent microscopic observations for spleen were changes in the size and number of germinal centers (58%), acidophilic (hyaline) material in lymphoid follicles (52%), and compound lymphoid follicles (20%). The most frequent microscopic observations in the MLN were eosinophil infiltrates (90%), changes in size and number of germinal centers (42%), and brown pigment (21%). The only meaningful relationships ( r2 > 0.3) were positive correlations between reticuloendothelial hyperplasia and malarial pigment in the spleen and between each of these observations and spleen weight parameters. We conclude that determination of test article-related effects on the immune system in routine monkey toxicology studies requires careful consideration and a weight-of-evidence approach due to the low numbers of animals/group, the inherent variability in spleen and MLN parameters, and the infrequent correlation among immune system-related end points.

Maturity-related Variability of the Thymus in Cynomolgus Monkeys (Macaca fascicularis).

Terminal body weights (TBWs), thymus weight parameters, and thymus morphology were retrospectively evaluated in 453 cynomolgus monkeys assigned to control groups on nonclinical toxicity studies. Morphology of bone, ovary, and testis/epididymis were used to determine maturity status of individual animals. There was no correlation between TBW and thymus weight (absolute and/or relative to TBW or brain weight). Thymus weight parameters and grades of decreased lymphocytes in the thymus were highly variable in immature animals compared to mature animals. There was also high (up to 11-fold) variability of thymus weight parameters within a given control group on the same study (generally 3 or 4 animals per sex). Several parameters evaluated had more pronounced age-related changes in males when compared to females. Our results demonstrate the inherent variability of thymus weight parameters and morphologic observations for cynomolgus monkeys on toxicology studies. Changes in thymus parameters in cynomolgus monkeys are unreliable indicators of immunomodulation or immunotoxicity in the absence of other relevant findings. Therefore, the thymus parameters commonly evaluated in preclinical safety assessments should not be the primary data set used to determine the presence of a direct test article-related effect on the immune system.

Development of 2-aminooxazoline 3-azaxanthenes as orally efficacious ß-secretase inhibitors for the potential treatment of Alzheimer's disease.

The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.

Retinal Toxicity Induced by a Novel ß-secretase Inhibitor in the Sprague-Dawley Rat.

ß-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase. The earliest detectable change in the retina was an increase in autofluorescent granules in the retinal pigment epithelium (RPE) on day 5; however, there were no treatment-related light microscopic changes observed in the neuroretina and no changes observed by fundus autofluorescence or routine ophthalmoscopic examination after 28 days of dosing. Following 2 months of recovery, there was significant retinal thinning attributed to loss of photoreceptor nuclei from the outer nuclear layer. Electroretinographic changes were observed as early as day 14, before any microscopic evidence of photoreceptor loss. BACE1 knockout rats were generated and found to have normal retinal morphology indicating that the retinal toxicity induced by AMG-8718 was likely off-target. These results suggest that AMG-8718 impairs phagolysosomal function in the rat RPE, which leads to photoreceptor dysfunction and ultimately loss of photoreceptors.

in silico transcriptome dissection of neocortical excitatory neurogenesis via joint matrix decomposition and transfer learning.

The rising quality and amount of multi-omic data across biomedical science demands that we build innovative solutions to harness their collective discovery potential. From publicly available repositories, we have assembled and curated a compendium of gene-level transcriptomic data focused on mammalian excitatory neurogenesis in the neocortex. This collection is open for exploration by both computational and cell biologists at nemoanalytics.org, and this report forms a demonstration of its utility. Applying our novel structured joint decomposition approach to mouse, macaque and human data from the collection, we define transcriptome dynamics that are conserved across mammalian excitatory neurogenesis and which map onto the genetics of human brain structure and disease. Leveraging additional data within NeMO Analytics via projection methods, we chart the dynamics of these fundamental molecular elements of neurogenesis across developmental time and space and into postnatal life. Reversing the direction of our investigation, we use transcriptomic data from laminar-specific dissection of adult human neocortex to define molecular signatures specific to excitatory neuronal cell types resident in individual layers of the mature neocortex, and trace their emergence across development. We show that while many lineage defining transcription factors are most highly expressed at early fetal ages, the laminar neuronal identities which they drive take years to decades to reach full maturity. Finally, we interrogated data from stem-cell derived cerebral organoid systems demonstrating that many fundamental elements of in vivo development are recapitulated with high-fidelity in vitro, while specific transcriptomic programs in neuronal maturation are absent. We propose these analyses as specific applications of the general approach of combining joint decomposition with large curated collections of analysis-ready multi-omics data matrices focused on particular cell and disease contexts. Importantly, these open environments are accessible to, and must be fueled with emerging data by, cell biologists with and without coding expertise.

Preservation of co-expression defines the primary tissue fidelity of human neural organoids.

Human neural organoid models offer an exciting opportunity for studying often inaccessible human-specific brain development; however, it remains unclear how precisely organoids recapitulate fetal/primary tissue biology. Here, we characterize field-wide replicability and biological fidelity through a meta-analysis of single-cell RNA-sequencing data for first and second trimester human primary brain (2.95 million cells, 51 datasets) and neural organoids (1.63 million cells, 130 datasets). We quantify the degree to which primary tissue cell-type marker expression and co-expression are recapitulated in organoids across 12 different protocol types. By quantifying gene-level preservation of primary tissue co-expression, we show neural organoids lie on a spectrum ranging from virtually no signal to co-expression near indistinguishable from primary tissue data, demonstrating high fidelity is within the scope of current methods. Additionally, we show neural organoids preserve the cell-type specific co-expression of developing rather than adult cells, confirming organoids are an appropriate model for primary tissue development. Overall, quantifying the preservation of primary tissue co-expression is a powerful tool for uncovering unifying axes of variation across heterogeneous neural organoid experiments.

Population variability in X-chromosome inactivation across 9 mammalian species.

One of the two X chromosomes in female mammals is epigenetically silenced in embryonic stem cells by X chromosome inactivation (XCI). This creates a mosaic of cells expressing either the maternal or the paternal X allele. The XCI ratio, the proportion of inactivated parental alleles, varies widely among individuals, representing the largest instance of epigenetic variability within mammalian populations. While various contributing factors to XCI variability are recognized, namely stochastic and/or genetic effects, their relative contributions are poorly understood. This is due in part to limited cross-species analysis, making it difficult to distinguish between generalizable or species-specific mechanisms for XCI ratio variability. To address this gap, we measured XCI ratios in nine mammalian species (9,143 individual samples), ranging from rodents to primates, and compared the strength of stochastic models or genetic factors for explaining XCI variability. Our results demonstrate the embryonic stochasticity of XCI is a general explanatory model for population XCI variability in mammals, while genetic factors play a minor role.

OncoVEXmGM-CSFexpands tumor antigen-specific CD8+ T-cell response in preclinical models.

BACKGROUND: Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEXmGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEXmGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. METHODS: A novel B16F10 syngeneic tumor model with both HSV-1-permissive subcutaneous tumors and HSV-1-refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEXmGM-CSF treatment alone or in combination with checkpoint inhibitors. RESULTS: Intratumoral injection of OncoVEXmGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEXmGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEXmGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. CONCLUSIONS: These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.

Variability of cross-tissue X-chromosome inactivation characterizes timing of human embryonic lineage specification events.

X-chromosome inactivation (XCI) is a random, permanent, and developmentally early epigenetic event that occurs during mammalian embryogenesis. We harness these features to investigate characteristics of early lineage specification events during human development. We initially assess the consistency of X-inactivation and establish a robust set of XCI-escape genes. By analyzing variance in XCI ratios across tissues and individuals, we find that XCI is shared across all tissues, suggesting that XCI is completed in the epiblast (in at least 6-16 cells) prior to specification of the germ layers. Additionally, we exploit tissue-specific variability to characterize the number of cells present during tissue-lineage commitment, ranging from approximately 20 cells in liver and whole blood tissues to 80 cells in brain tissues. By investigating the variability of XCI ratios using adult tissue, we characterize embryonic features of human XCI and lineage specification that are otherwise difficult to ascertain experimentally.

Strategies to evaluate potential effector function of glycan variants: a case study of ordesekimab (AMG 714 or PRV-015).

The potential for effector functions of therapeutic antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), is a biological activity of interest for characterization, regardless of if ADCC is an intended primary pharmacological effect. The composition of the conserved antibody Fc glycan can vary as a function of post-translational processing which may affect the binding affinity to Fc receptors, leading to a change of effector activity. Ordesekimab (AMG 714 or PRV-015), a fully human immunoglobulin G1-kappa anti-interleukin (IL)-15 monoclonal antibody, is in clinical development for celiac disease. The binding of ordesekimab to IL-15 inhibits the interaction of IL-15 with the IL-2Rß and common γ chain of the IL-15 receptor complex, but not with the IL-15Rα chain. Therefore, the simultaneous binding of ordesekimab to the Fcγ receptor (R) IIIα expressed on natural killer (NK) cells and to the IL-15/IL-15Rα complex on cells such as monocytes may theoretically enable ADCC toward the IL-15Rα-expressing cells. The high mannose (HM) levels on the Fc glycan were found to vary in different lots of ordesekimab resulting from refinements to the manufacturing process, and the impact on ordesekimab-mediated ADCC activity was evaluated in in vivo and in vitro studies. A review of nonclinical and clinical data found no evidence of ordesekimab-induced depletion of monocytes, or cytotoxicity in organs with wide IL-15Rα expression, suggesting a lack of in vivo ADCC activity. In addition, in vitro peripheral blood mononuclear cells-based ADCC assay did not reveal any cytolytic effect of ordesekimab with various levels of HM content when cocultured with recombinant human IL-15. Taken together, these data demonstrate that ADCC is not a potential liability for ordesekimab and does not contribute to the reduction of IL-15-mediated inflammation, the intended pharmacological effect.

Hallmarks of primary neurulation are conserved in the zebrafish forebrain.

Primary neurulation is the process by which the neural tube, the central nervous system precursor, is formed from the neural plate. Incomplete neural tube closure occurs frequently, yet underlying causes remain poorly understood. Developmental studies in amniotes and amphibians have identified hingepoint and neural fold formation as key morphogenetic events and hallmarks of primary neurulation, the disruption of which causes neural tube defects. In contrast, the mode of neurulation in teleosts has remained highly debated. Teleosts are thought to have evolved a unique mode of neurulation, whereby the neural plate infolds in absence of hingepoints and neural folds, at least in the hindbrain/trunk where it has been studied. Using high-resolution imaging and time-lapse microscopy, we show here the presence of these morphological landmarks in the zebrafish anterior neural plate. These results reveal similarities between neurulation in teleosts and other vertebrates and hence the suitability of zebrafish to understand human neurulation.