Embryonic exposure to flubendiamide induces hepatotoxicity in domestic chicks by altering drug-metabolizing enzymes, antioxidant status, and liver function.

Pesticides have increased crop yield but severely impacted ecosystems and non-target organisms. Flubendiamide, a new generation pesticide, targets insect larvae but also affects non-target organisms. This study examines the effects of lowest observed effect concentration of technical grade flubendiamide (0.5 µg/µL) flubendiamide on chick liver, focusing on cytochrome P450 (CYP) enzyme expression, oxidative stress, and liver damage. Chick embryos treated with flubendiamide showed significant alterations in CYP mRNA and protein levels, indicating increased toxicant accumulation. Elevated CYP3A4, CYP1A1, CYP1A2, and CYP2C19 levels were noted, suggesting enhanced biotransformation and detoxification processes. However, increased oxidative byproducts led to oxidative stress, as evidenced by decreased glutathione (GSH) levels and elevated superoxide dismutase (SOD) and catalase activities. DCFDA staining confirmed increased hydrogen peroxide (H2O2) levels, indicating heightened reactive oxygen species (ROS). Liver function tests revealed significant increases in serum ALP, ALT, and AST levels, indicating acute liver damage. Histopathological analysis showed structural liver damage, including expanded sinusoidal spaces, impaired portal veins, and compromised hepatocyte architecture. These findings underscore flubendiamide's potential hepatotoxicity in non-target organisms, emphasizing the need for cautious pesticide use to minimize environmental impacts.

A Modified Quadrature Birdcage Coil Incorporated With a Curved Feature for In Ovo MR Imaging.

Goal: This study presents a novel MRI coil design approach explicitly tailored for chick embryo measurements, with the primary objective of improving sensitivity and coverage. Methods: The limitations posed by conventional birdcage coils were addressed by introducing a curvature feature into a standard coil. The performance of the modified coil was assessed using EM simulations and experimental evaluations, which were subsequently validated using a 7 T MRI scanner. A comparative analysis was conducted against a standard quadrature low-pass birdcage coil to evaluate key factors. Results: The proposed coil demonstrated improved SNR and uniformity, particularly in the proximity of the end-rings. These results were consistent with the findings obtained from the simulations. Conclusions: The use of our innovative birdcage coil design holds promise and offers practical potential for in ovo studies.

Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation.

Neural Crest cells (NC) are a multipotent cell population that give rise to a multitude of cell types including Schwann cells (SC) in the peripheral nervous system (PNS). Immature SC interact with neuronal axons via the neuregulin 1 (NRG1) ligand present on the neuronal surface and ultimately form the myelin sheath. Multiple attempts to derive functional SC from pluripotent stem cells have met challenges with respect to expression of mature markers and axonal sorting. Here, they hypothesized that sustained signaling from immobilized NRG1 (iNRG1) might enhance the differentiation of NC derived from glabrous neonatal epidermis towards a SC phenotype. Using this strategy, NC derived SC expressed mature markers to similar levels as compared to explanted rat sciatic SC. Signaling studies revealed that sustained NRG1 signaling led to yes-associated protein 1 (YAP) activation and nuclear translocation. Furthermore, NC derived SC on iNRG1 exhibited mature SC function as they aligned with rat dorsal root ganglia (DRG) neurons in an in vitro coculture model; and most notably, aligned on neuronal axons upon implantation in a chick embryo model in vivo. Taken together their work demonstrated the importance of signaling dynamics in SC differentiation, aiming towards development of drug testing platforms for de-myelinating disorders.

Assessment of efficacy of chrysin in diabetes-associated cardiac complications in chick embryo and murine model.

OBJECTIVES: Patients with type 2 diabetes or prolonged diabetic condition are webbed into cardiac complications. This study aimed to ascertain the utility of chick embryo as an alternative to the mammalian model for type 2 diabetes-induced cardiac complications and chrysin as a protective agent. METHODS: Diabetes was activated in ovo model (chick embryo) using glucose along with ß-hydroxybutyric acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Alamar, and Kenacid blue assay were used to compare with chrysin-administered group. Blood glucose level, total cholesterol, triglyceride, and high-density lipoprotein were considered as endpoints. Diabetes was induced in Wistar albino rats by administering a high-fat diet and a subdued dose of streptozotocin (35 mg/kg, b.w). Percentage of glycated hemoglobin, creatinine kinase-MB, tumor necrosis factor-α, and C-reactive protein were evaluated and compared with chrysin administered group. KEY FINDINGS: Chrysin treatment improved elevated blood glucose levels and dyslipidemia in a diabetic group of whole embryos. Condensed cellular growth and protein content as well as enhanced cytotoxicity in ovo were shielded by chrysin. Chrysin reduced cardiac and inflammatory markers in diabetic rats and provided cellular protection to damage the heart of diabetic rats. CONCLUSION: The protective action of chrysin in ovo model induced a secondary complication associated with diabetes, evidenced that the ovo model is an effective alternative in curtailing higher animal use in scientific research.

Single cell RNA-sequencing and RNA-tomography of the avian embryo extending body axis.

Introduction: Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord-forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing. Methods: Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo. Results and Discussion: Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNA-tomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.

Hemodynamics During Development and Postnatal Life.

A well-developed heart is essential for embryonic survival. There are constant interactions between cardiac tissue motion and blood flow, which determine the heart shape itself. Hemodynamic forces are a powerful stimulus for cardiac growth and differentiation. Therefore, it is particularly interesting to investigate how the blood flows through the heart and how hemodynamics is linked to a particular species and its development, including human. The appropriate patterns and magnitude of hemodynamic stresses are necessary for the proper formation of cardiac structures, and hemodynamic perturbations have been found to cause malformations via identifiable mechanobiological molecular pathways. There are significant differences in cardiac hemodynamics among vertebrate species, which go hand in hand with the presence of specific anatomical structures. However, strong similarities during development suggest a common pattern for cardiac hemodynamics in human adults. In the human fetal heart, hemodynamic abnormalities during gestation are known to progress to congenital heart malformations by birth. In this chapter, we discuss the current state of the knowledge of the prenatal cardiac hemodynamics, as discovered through small and large animal models, as well as from clinical investigations, with parallels gathered from the poikilotherm vertebrates that emulate some hemodynamically significant human congenital heart diseases.

Effect of aripiprazole on neural tube development in early chick embryos.

INTRODUCTION: Aripiprazole (ARI) is a recently developed antipsychotic medication that belongs to the second generation of antipsychotics. The literature has contradictory information regarding ARI, which has been classified as pregnant use category C by the FDA. METHODS: 125 pathogen-free fertilized eggs were incubated for 28 h and divided into five groups of 25 eggs each (including the control group), and 18 eggs with intact integrity were selected from each group. After the experimental groups were divided, ARI was administered subblastodermally with a Hamilton micro-injector at 4 different doses (1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg). At the 48th hour of incubation, all eggs were hatched and embryos were removed from the embryonic membranes. And then morphologic (position of the neural tube (open or closed), crown-rump length, number of somites, embryological development status), histopathologic (apoptosis (caspase 3), cell proliferation (PCNA), in situ recognition of DNA breaks (tunnel)), genetic (BRE gene expression) analyzes were performed. RESULTS: According to the results of the morphological analysis, when the frequency of neural tube patency was evaluated among the experimental groups, a statistically significant difference was determined between the control group and all groups (p < 0.001). In addition, the mean crown-rump length and somite number of the embryos decreased in a dose-dependent manner compared to the control group. It was determined that mRNA levels of the BRE gene decreased in embryos exposed to ARI compared to the control group (p < 0.001). CONCLUSION: Morphologically, histopathologically, and genetically, aripiprazole exposure delayed neurogenesis and development in early chick embryos. These findings suggest its use in pregnant women may be teratogenic. We note that these results are preliminary for pregnant women, but they should be expanded and studied with additional and other samples.

Effect of post-gastrulation exposure to acrylamide on chick embryonic development.

The critical developmental stages of the embryo are strongly influenced by the dietary composition of the mother. Acrylamide is a food contaminant that can form in carbohydrate-rich foods that are heat-treated. The aim of this study was to investigate the toxicity of a relatively low dose of acrylamide on the development of the neural tube in the early stage chick embryos. Specific pathogen-free fertilized eggs (n = 100) were treated with acrylamide (0.1, 0.5, 2.5, 12.5 mg/kg) between 28-30th hours of incubation and dissected at 48th hours. In addition to morphological and histopathological examinations, proliferating cell nuclear antigen (PCNA) and caspase 3 were analyzed immunohistochemically. The brain and reproductive expression gene (BRE) was analyzed by RT-PCR. Acrylamide exposure had a negative effect on neural tube status even at a very low dose (0.1 mg/kg) (p < 0.05). Doses of 0.5 mg/kg and above caused a delay in neural tube development (p < 0.05). Crown-rump length and somite count decreased dose-dependently, while this decrease was not significant in the very low dose group (p > 0.05), which was most pronounced at doses of 2.5 and 12.5 mg/kg (p < 0.001). Acrylamide exposure dose-dependently decreased PCNA and increased caspase 3, with this change being significant at doses of 0.5 mg/kg and above (p < 0.001). BRE was downregulated at all acrylamide exposures except in the very low dose group (0.1 mg/kg). In conclusion, we find that acrylamide exposure (at 0.5 mg/kg and above) in post-gastrulation delays neural tube closure in chicken embryos by suppressing proliferation and apoptosis induction and downregulating BRE gene expression.

Isolation and Characterization of Extracellular Vesicles of Chick Embryo Blood.

Exosomes from plants or animals are a cheap, available, and promising option in medicine, which can be used for the detection or treatment of various diseases. This study aims to evaluate the antitoxic and antioxidant properties of Extracellular vesicle (EVs) extracted from chicken embryo blood using a fibroblast cell line (NIH/3T3). EVs from chick embryos were extracted in this experimental investigation using the sedimentation method and examined using dynamic light scattering (DLS) and field emission electron microscopy (FE-SEM). The protein concentration and overall antioxidant capacity of the EVs were determined using bicinchoninic acid (BCA) and antioxidant capacity (FRAP). EVs were added to NIH/3T3 cells at varying concentrations (1, 2, and 10 mg/ml), and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay test was used to measure cell survival. The size of the isolated EVs was confirmed to be less than 100 nm by electron microscopy and DLS. The quantity of protein in these EVs was 3200 µg/ml, and their total antioxidant capacities were 3130.17, 1914.122, and 976.9 µMol/L. The MTT test findings demonstrated that NIH/3T3 cells survived treatment with EVs (P ≤ 0.001) compared to the control group. Antioxidant-rich and protein-rich exosomes in chicken embryos may be valuable in managing oxidative stress.

An optimized CT-dense agent perfusion and micro-CT imaging protocol for chick embryo developmental stages.

Compared to classical techniques of morphological analysis, micro-CT (µ-CT) has become an effective approach allowing rapid screening of morphological changes. In the present work, we aimed to provide an optimized micro-CT dense agent perfusion protocol and µ-CT guidelines for different stages of chick embryo cardiogenesis. Our study was conducted over a period of 10 embryonic days (Hamburger-Hamilton HH36) in chick embryo hearts. During the perfusion of the micro-CT dense agent at different developmental stages (HH19, HH24, HH27, HH29, HH31, HH34, HH35, and HH36), we demonstrated that durations and volumes of the injected contrast agent gradually increased with the heart developmental stages contrary to the flow rate that was unchanged during the whole experiment. Analysis of the CT imaging confirmed the efficiency of the optimized parameters of the heart perfusion.

Hox-A2 protein expression in avian jaws cartilages and muscle primordia development.

OBJECTIVE: To elucidate the branchial origin of the articular and the square (homology of the malleus and the incus of mammals), we used immunohistochemistry to analyse the expression of the Hox-A2 protein during cephalogenesis in chickens. MATERIALS AND METHODS: Immunohistochemistry on paraffin sections of embryos from stage HH16 to HH40. RESULTS: In addition to the columella (equivalent to the mammalian stapes), the joint between the articular and the quadrate bones, and the retro-articular process of the articular (homologous to the short process of the malleus) express Hox-A2, suggesting an intervention of the 2nd arch in their formation. However, we fortuitously observed very intense expression within the early muscle plate of the second arch, which then generalized to all cephalic muscles, and extended to the trunk's myotomes. In the cartilage, the presence of the protein disappeared at stage 35. DISCUSSION AND CONCLUSION: The present results, while confirming the contribution of the second arch to the development of avian equivalents of the mammalian ear ossicles, strongly suggest that the Hox-A2 gene plays a role in muscle development, which remains to be elucidated by more sophisticated techniques.

Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

Current research on mechanisms of limb bud development, and challenges for the next decade.

The developmental mechanisms of limb buds have been studied in developmental biology as an excellent model of pattern formation. Chick embryos have contributed to the discovery of new principles in developmental biology, as it is easy to observe live embryos and manipulate embryonic tissues. Herein, I outline recent findings and future issues over the next decade regarding three themes, based on my research: limb positioning, proximal-distal limb elongation and digit identity determination. First, how hindlimb position is determined at the molecular level is described, with a focus on the transforming growth factor-ß signaling molecule GDF11. Second, I explain how the cell population in the limb bud deforms with developmental progress, shaping the limb bud with elongation along the proximal-distal axis. Finally, I describe the developmental mechanisms that determine digit identity through the interdigits.

In a century from agitated cells to human organoids.

Reaching back more than a century, suspension cultures have provided major insights into processes of histogenesis; e.g., cell communication, distinction of self/nonself, cell sorting and cell adhesion. Besides studies on lower animals, the vertebrate retina served as excellent reaggregate model to analyze 3D reconstruction of a complex neural laminar tissue. Methodologically, keeping cells under suspension is essential to achieve tissue organisation in vitro; thereby, the environmental conditions direct the emergent histotypic particulars. Recent progress in regenerative medicine is based to a large extent on human induced pluripotent stem cells (hiPSCs), which are cultured under suspension. Following their genetically directed differentiation into various histologic 3D structures, organoids provide excellent multipurpose in vitro assay models, as well as tissues for repair transplantations. Historically, a nearly fully laminated retinal spheroid from avian embryos was achieved already in 1984, foreshadowing the potential of culturing stem cells under suspension for tissue reconstruction purposes.

Pomegranate extract in polyphosphate-fluoride mouthwash reduces enamel demineralization.

OBJECTIVES: To evaluate the anti-demineralizing effect of a mouthwash comprising pomegranate peel extract (PPE 3%), sodium trimetaphosphate (TMP 0.3%), and fluoride (F 225 ppm) in an in situ study, and to assess its irritation potential in an ex vivo study. METHODS: This double-blind crossover study was conducted in four phases with 7 days each. Twelve volunteers used palatal appliances containing enamel blocks, which were subjected to cariogenic challenges. The ETF formulation (PPE + TMP + F, pH 7.0), TF formulation (TMP + F, pH 7.0), deionized water (W, pH 7.0), and essential oil commercial mouthwash (CM, 220 ppm F, pH 4.3) were dropped onto the enamel twice daily. The percentage of surface hardness loss, integrated loss of subsurface hardness, calcium, phosphorus, and fluoride in enamel and biofilms were determined. In addition, alkali-soluble extracellular polysaccharide concentrations were analyzed in the biofilms. The irritation potential was evaluated using the hen's egg chorioallantoic membrane test through the vascular effect produced during 300-s of exposure. RESULTS: ETF was the most efficacious in preventing demineralization. It also showed the highest concentrations of calcium and phosphorus in the enamel and in the biofilm, as well as the lowest amount of extracellular polysaccharides in the biofilm. In the eggs, ETF produced light reddening, whereas CM led to hyperemia and hemorrhage. CONCLUSIONS: The addition of PPE to formulations containing TMP and F increased its anti-demineralizing property, and this formulation presented a lower irritation potential than the CM. CLINICAL RELEVANCE: ETF can be a promising alternative alcohol-free mouthwash in patients at high risk of caries.

DLX gene expression in the developing chick pharyngeal arches and relationship to endothelin signaling and avian jaw patterning.

BACKGROUND: A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest-derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage-specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. RESULTS: Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down-regulation of ventral-specific DLX genes and confirms their functional role in avian jaw patterning. CONCLUSIONS: This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX-regulated genes in the chick.

Embryonic development of the chick pineal gland throughout the incubation periods.

Birds have a very different pineal gland structure morphologically and cytologically. The structure of the organ shows significant changes during the incubation periods. This study, which follows the embryological development of the pineal gland and makes histomorphometric measurements of the cellular elements that make up the gland parenchyma, is a current reference for studies in these areas. These brains were taken from 24 Babcock White Leghorn chick embryos on the 10th, 13th, 16th and 21st days of incubation. At 10th embryonic day, the pineal recess was in the structure of an elongated pineal canal. Solid rosette-shaped cell clusters were transforming into round vesicles with a small lumen. These vesicles had developed into larger, oval-shaped follicles with a well-defined central lumen. On 13th day, it was observed that the number and development of follicles increased considerably. The pineal gland showed a follicular-solid structure in 16th day embryos. While the mean follicle diameter was determined as 123.46 ± 13.28 µm on the 10th embryonic day, the highest value was measured as 187.62 ± 7.37 µm on the hatching day (p < 0.05). While the mean follicle area had the lowest value in the 10th day embryos, it was determined that this value gradually increased compared to the advancing embryonic days (p < 0.05). As conclusion, it is thought that this study provides new data to the literature about pineal gland development by monitoring the histological and histomorphometric developments of chick pineal gland in different incubation periods.

Observation of the neuroprotective efficacy of vitamin K in a streptozocin-induced diabetes model in chick embryos.

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia due to insulin deficiency and/or resistance. Vitamin K (VK) is a group of fat-soluble molecules, including naturally occurring vitamin K1 (phylloquinone). vitamin K2 (menaquinone), and synthetic vitamin K3 (menadione). Beyond coagulation, the health benefits of VK have been described to play different roles in both physiological and pathological processes such as inflammation, energy metabolism, neuroprotection, cellular growth, and survival. It was aimed to observe the antioxidant and/or neuroprotective activity of vitamin K1 in our model of chick embryo diabetic neuropathy (DN) induced by streptozotocin (STZ). Ninety White Leghorn, fertile and 0-day-old SPF (specific pathogen-free) eggs (57 ± 4 gr) were used in the study. Chick embryo blood brain tissues were taken for biochemical evaluation. Plasma insulin and glucose levels were measured. In addition, brain tissue total antioxidant level (TAS), total oxidant level (TOS), malondialdehyde (MDA), and vascular endothelial growth factor (VEGF) levels were measured. Plasma glucose levels were higher in the STZ-treated groups and lower in the treatment groups. Plasma insulin levels were observed to be higher in STZ groups in groups treated with high VK. Low TAS, high MDA, TOS, and VEGF levels were recorded in brain tissue STZ groups. Low VEGF, TOS, and MDA levels were recorded in the group treated with the highest VK, while high TAS levels were observed. In our STZ-induced chick embryo diabetic neuropathy model, we observed that VK1 reduced oxidant damage by showing antioxidant properties or by modulating antioxidant enzymes.

Effects of the Herbal Product Curcumin on Cardiomyocytes in Micromass Culture and the Potential Role It May Play in Pregnancy and Development.

Introduction Herbal medicine (HM) consumption during pregnancy has been on the rise in many parts of the world. Curcumin is a proven antioxidant and anti-inflammatory herb component, having demonstrated efficacy in alleviating various diseases. However, there is conflicting evidence with regards to its effect on pregnancy. We assess the safety profile of the main component of turmeric, curcumin, during pregnancy. Furthermore, to investigate curcumin in combination with known teratogen ethanol to identify any protective effect that curcumin might exert. Method Embryonic chick cardiomyocytes in micromass culture were treated with varying concentrations of curcumin. Three endpoints were used to determine the effect of curcumin on these cells: contractile activity (morphological score), cell viability (resazurin assay), and total protein content (kenacid blue assay). Results Curcumin demonstrated cytotoxicity at the highest tested concentrations (10-20µM) by significantly reducing cell activity and total protein. The results of morphological scoring suggest that repeated investigations would have revealed the teratogenic potential of curcumin. Lower concentrations (50nM) of curcumin were comparable to the control. The combination of a non-toxic concentration of curcumin with ethanol revealed additive toxicity. Conclusion It seems unlikely that curcumin will adversely affect the embryo at low doses due to issues of bioavailability. The findings of cytotoxicity and possible teratogenicity at high concentrations are a concern. Due to the limited information available regarding curcumin metabolism in human embryos, advancements in curcumin delivery systems, and the high likelihood of overconsumption, further in vivo research using animal models is required.

Accurate staging of chick embryonic tissues via deep learning of salient features.

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.