Nematode eggshells: A new anatomical and terminological framework, with a critical review of relevant literature and suggested guidelines for the interpretation and reporting of eggshell imagery.

A literature review for a recent ultrastructural study of a trichinelloid eggshell revealed consistently occurring errors in the literature on nematode eggshell anatomy. Examples included nematodes of medical, veterinary, and agricultural importance in several orders. Previous researchers had warned of some of these errors decades ago, but a comprehensive solution was not offered until 2012 when a clarifying new anatomical and developmental interpretation of nematode eggshells was proposed by members of the Caenorhabditis elegans Research Community. However, their findings were explained using arcane acronyms and technical jargon intended for an audience of experimental molecular geneticists, and so their papers have rarely been cited outside the C. elegans community. Herein we (1) provide a critical review of nematode eggshell literature in which we correct errors and relabel imagery in important historical reports; (2) describe common reporting errors and their causes using language familiar to researchers having a basic understanding of microscopy and nematode eggs; (3) recommend a new hexalaminar anatomical and terminological framework for nematode eggshells based on the 2012 C. elegans framework; and (4) recommend new unambiguous terms appropriate for the embryonated/larvated eggs regularly encountered by practicing nematodologists to replace ambiguous or ontogenetically restricted terms in the 2012 C. elegans framework. We also (5) propose a resolution to conflicting claims made by the C. elegans team versus classical literature regarding Layer #3, (6) extend the C. elegans hexalaminar framework to include the polar plugs of trichinelloids, and (7) report new findings regarding trichinelloid eggshell structure.

Title: La coque des œufs des nématodes : un nouveau cadre anatomique et terminologique, avec une revue critique de la littérature pertinente et des lignes directrices suggérées pour l'interprétation et la communication de l'imagerie des coques des œufs. Abstract: Une revue de la littérature pour une étude ultrastructurale récente de la coque de l'œuf d'un trichinelloïde a révélé des erreurs récurrentes dans la littérature sur l'anatomie de la coque de l'œuf des nématodes. Les exemples comprenaient des nématodes d'importance médicale, vétérinaire et agricole dans plusieurs ordres. Des chercheurs avaient mis en garde contre certaines de ces erreurs il y a des décennies, mais une solution complète n'a été proposée qu'en 2012, lorsqu'une nouvelle interprétation anatomique et développementale clarifiant la structure des coques des œufs de nématodes a été proposée par des membres de la communauté de recherche de Caenorhabditis elegans. Cependant, leurs découvertes ont été expliquées à l'aide d'acronymes mystérieux et d'un jargon technique destiné à un public de généticiens moléculaires expérimentaux, et leurs articles ont donc rarement été cités en dehors de la communauté de C. elegans. Ici, nous (1) fournissons une revue critique de la littérature sur les coques des œufs de nématodes dans laquelle nous corrigeons les erreurs et réétiquetons les images dans des rapports historiques importants; (2) décrivons les erreurs de description courantes et leurs causes en utilisant un langage familier aux chercheurs ayant une compréhension de base de la microscopie et des œufs de nématodes; (3) recommandons un nouveau cadre anatomique et terminologique hexalaminaire pour les coques des œufs de nématodes basé sur le cadre de C. elegans de 2012; et (4) recommandons de nouveaux termes non ambigus appropriés pour les œufs embryonnés/larvés régulièrement rencontrés par les spécialistes de nématodes en exercice pour remplacer les termes ambigus ou à restriction ontogénétique dans le cadre de C. elegans de 2012. Nous proposons également (5) une résolution des affirmations contradictoires de l'équipe C. elegans par rapport à la littérature classique concernant la couche 3, (6) étendons le cadre hexalaminaire de C. elegans pour inclure les bouchons polaires des trichinelloïdes, et (7) signalons de nouvelles découvertes concernant la structure de la coque des œufs des trichinelloïdes.

Organization of the body wall in lampreys informs the evolution of the vertebrate paired appendages.

Vertebrate paired appendages are one of the most important evolutionary novelties in vertebrates. During embryogenesis, the skeletal elements of paired appendages differentiate from the somatic mesoderm, which is a layer of lateral plate mesoderm. However, the presence of the somatic mesoderm in the common ancestor of vertebrates has been controversial. To address this problem, it is necessary but insufficient to understand the developmental process of lateral plate mesoderm formation in lamprey (jawless vertebrates) embryos. Here, I show the presence of the somatic mesoderm in lamprey (Lethenteron camtschaticum) embryos using plastic sectioning and transmission electron microscopy analysis. During the early pharyngeal stages, the somatic mesoderm transforms from the lateral plate mesoderm in the trunk region. Soon after, when the cardiac structures were morphologically distinct, the somatic mesoderm was recognized through the cardiac to more caudal regions. These findings indicated that the somatic mesoderm evolved before the emergence of paired appendages. I also discuss the developmental changes in the body wall organization in the common ancestor of vertebrates, which is likely related to the evolution of the paired appendages.

CEP97 phosphorylation by Dyrk1a is critical for centriole separation during multiciliogenesis.

Proper cilia formation in multiciliated cells (MCCs) is necessary for appropriate embryonic development and homeostasis. Multicilia share many structural characteristics with monocilia and primary cilia, but there are still significant gaps in our understanding of the regulation of multiciliogenesis. Using the Xenopus embryo, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase. Knockdown of either CEP97 or Dyrk1a disrupts cilia formation and centriole disengagement in MCCs, but this defect is rescued by overexpression of Separase. Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

E-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1's formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

Metal ion fluxes controlling amphibian fertilization.

Mammalian oocytes undergo major changes in zinc content and localization to be fertilized, the most striking being the rapid exocytosis of over 10 billion zinc ions in what are known as zinc sparks. Here, we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese. Synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through electron-nuclear double-resonance studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs, but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Canonical Wnt/ß-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with ß-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

Cryopreservation method for Drosophila melanogaster embryos.

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.

Ultrastructural studies of developing egg tooth in grass snake Natrix natrix (Squamata, Serpentes) embryos, supported by X-ray microtomography analysis.

The egg tooth development is similar to the development of all the other vertebrate teeth except earliest developmental stages because the egg tooth develops directly from the oral epithelium instead of the dental lamina similarly to null generation teeth. The developing egg tooth of Natrix natrix changes its curvature differently than the egg tooth of the other investigated unidentates due to the presence of the rostral groove. The developing grass snake egg tooth comprises dental pulp and the enamel organ. The fully differentiated enamel organ consists of outer enamel epithelium, stellate reticulum, and ameloblasts in its inner layer. The enamel organ directly in contact with the oral cavity is covered with periderm instead of outer enamel epithelium. Stellate reticulum cells in the grass snake egg tooth share intercellular spaces with the basal part of ameloblasts and are responsible for their nutrition. Ameloblasts during egg tooth differentiation pass through the following stages: presecretory, secretory, and mature. The ameloblasts from the grass snake egg tooth show the same cellular changes as reported during mammalian amelogenesis but are devoid of Tomes' processes. Odontoblasts of the developing grass snake egg tooth pass through the following classes: pre-odontoblasts, secretory odontoblasts, and ageing odontoblasts. They have highly differentiated secretory apparatus and in the course of their activity accumulate lipofuscin. Grass snake odontoblasts possess processes which are poor in organelles. In developing egg tooth cilia have been identified in odontoblasts, ameloblasts and cells of the stellate reticulum. Dental pulp cells remodel collagen matrix during growth of the grass snake egg tooth. They degenerate in a way previously not described in other teeth.

Telocytes are major constituents of the angiogenic apparatus.

The current study investigated role of telocytes (TCs) in angiogenesis during embryonic development of quail using immunohistochemistry (IHC), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The angiogenic apparatus consisted of TCs, endothelial cells, and macrophages. TCs were identified morphologically by their telopodes and podoms using TEM and SEM and immunohistochemically using CD34, and vascular endothelial growth factor (VEGF). TCs also expressed CD68. TCs formed a three-dimensional network and established direct contact with blood vessels, sprouting endothelial cells, and active macrophages, while exerting their effect through paracrine signaling. VEGF was also expressed by endothelial cells and macrophages. Matrix metalloproteinase-9 (MMP-9) was expressed by TCs, endothelial cells, and macrophages. In conclusion, the expression of VEGF by TCs, endothelial cells, and macrophages is required for the proliferation and migration of endothelial cells and vascular growth. The expression of MMP-9 by TCs, endothelial cells, and macrophages is essential for the degradation of extracellular matrix (ECM) components during neoangiogenesis. Macrophages may facilitate phagocytosis and elimination of the degraded ECM components.

Exceptionally preserved early Cambrian bilaterian developmental stages from Mongolia.

Fossilized invertebrate embryonic and later developmental stages are rare and restricted largely to the Ediacaran-Cambrian, providing direct insight into development during the emergence of animal bodyplans. Here we report a new assemblage of eggs, embryos and bilaterian post-embryonic developmental stages from the early Cambrian Salanygol Formation of Dzhabkan Microcontinent of Mongolia. The post-embryonic developmental stages of the bilaterian are preserved with cellular fidelity, possessing a series of bilaterally arranged ridges that compare to co-occurring camenellan sclerites in which the initial growth stages retain the cellular morphology of modified juveniles. In this work we identify these fossils as early post-embryonic developmental stages of camenellans, an early clade of stem-brachiopods, known previously only from isolated sclerites. This interpretation corroborates previous reconstructions of camenellan scleritomes with sclerites arranged in medial and peripheral concentric zones. It further supports the conjecture that molluscs and brachiopods are descended from an ancestral vermiform and slug-like bodyplan.

Structural and ultrastructural studies on the developing vomeronasal sensory epithelium in the grass snake Natrix natrix (Squamata: Colubroidea).

The sensory olfactory epithelium and the vomeronasal sensory epithelium (VSE) are characterized by continuous turnover of the receptor cells during postnatal life and are capable of regeneration after injury. The VSE, like the entire vomeronasal organ, is generally well developed in squamates and is crucial for detection of pheromones and prey odors. Despite the numerous studies on embryonic development of the VSE in squamates, especially in snakes, an ultrastructural analysis, as far as we know, has never been performed. Therefore, we investigated the embryology of the VSE of the grass snake (Natrix natrix) using electron microscopy (SEM and TEM) and light microscopy. As was shown for adult snakes, the hypertrophied ophidian VSE may provide great resolution of changes in neuron morphology located at various epithelial levels. The results of this study suggest that different populations of stem/progenitor cells occur at the base of the ophidian VSE during embryonic development. One of them may be radial glia-like cells, described previously in mouse. The various structure and ultrastructure of neurons located at different parts of the VSE provide evidence for neuronal maturation and aging. Based on these results, a few nonmutually exclusive hypotheses explaining the formation of the peculiar columnar organization of the VSE in snakes were proposed.

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy.

Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.

Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

Developmental stages of zebrafish (Danio rerio) embryos and toxicological studies using foldscope microscope.

Zebrafish (Danio rerio), is a well-established vertebrate animal model widely used in developmental biology and toxicological research. In the present study, foldscope is used as an innovative tool to study the developmental stages and toxicological analysis of the zebrafish embryos. Briefly, the developmental stages, such as zygote, cleavage, blastula, gastrula, segmentation, and pharyngula formation are observed and documented using simple foldscope. Toxicological parameters upon exposure to different concentration of ethanol extract of Curcuma longa and its lead compound, ar-turmerone along with rhodamine B (bio-coupler) on zebrafish embryos are analyzed upto 72 hr using foldscopes in live condition. The lethal endpoints, such as coagulation, lack of somite formation, non-detachment of tail, and lack of heartbeat are clearly monitored and documented using foldscope. Bio-evaluation of test compounds with the aid of foldscope confirms that the toxicity is directly proportional to the concentration. Our results conclude that, ethanol extract of C. longa, ar-turmerone and rhodamine B exposed embryos remains healthy up to 96, 48, and 24 µg concentrations, respectively. Embryos exposed to higher concentrations become coagulated, however normal physiological active movement of tail lashing and heartbeat are evident in lower concentration exposed embryos. Except coagulation, no other abnormalities are observed and interestingly, the hatching ability is not delayed, when compared with the control embryos. It is confirmed that the test compounds are not highly toxic to zebrafish embryos. Hence it can be used for further analysis, especially for studying the neural-regeneration and its neuronal development in zebrafish embryos.

The denticle surface of thresher shark tails: Three-dimensional structure and comparison to other pelagic species.

Shark skin denticles (scales) are diverse in morphology both among species and across the body of single individuals, although the function of this diversity is poorly understood. The extremely elongate and highly flexible tail of thresher sharks provides an opportunity to characterize gradients in denticle surface characteristics along the length of the tail and assess correlations between denticle morphology and tail kinematics. We measured denticle morphology on the caudal fin of three mature and two embryo common thresher sharks (Alopias vulpinus), and we compared thresher tail denticles to those of eleven other shark species. Using surface profilometry, we quantified 3D-denticle patterning and texture along the tail of threshers (27 regions in adults, and 16 regions in embryos). We report that tails of thresher embryos have a membrane that covers the denticles and reduces surface roughness. In mature thresher tails, surfaces have an average roughness of 5.6 µm which is smoother than some other pelagic shark species, but similar in roughness to blacktip, porbeagle, and bonnethead shark tails. There is no gradient down the tail in roughness for the middle or trailing edge regions and hence no correlation with kinematic amplitude or inferred magnitude of flow separation along the tail during locomotion. Along the length of the tail there is a leading-to-trailing-edge gradient with larger leading edge denticles that lack ridges (average roughness = 9.6 µm), and smaller trailing edge denticles with 5 ridges (average roughness = 5.7 µm). Thresher shark tails have many missing denticles visible as gaps in the surface, and we present evidence that these denticles are being replaced by new denticles that emerge from the skin below.

Lamina-Dependent Stretching and Unconventional Chromosome Compartments in Early C. elegans Embryos.

Current models suggest that chromosome domains segregate into either an active (A) or inactive (B) compartment. B-compartment chromatin is physically separated from the A compartment and compacted by the nuclear lamina. To examine these models in the developmental context of C. elegans embryogenesis, we undertook chromosome tracing to map the trajectories of entire autosomes. Early embryonic chromosomes organized into an unconventional barbell-like configuration, with two densely folded B compartments separated by a central A compartment. Upon gastrulation, this conformation matured into conventional A/B compartments. We used unsupervised clustering to uncover subpopulations with differing folding properties and variable positioning of compartment boundaries. These conformations relied on tethering to the lamina to stretch the chromosome; detachment from the lamina compacted, and allowed intermingling between, A/B compartments. These findings reveal the diverse conformations of early embryonic chromosomes and uncover a previously unappreciated role for the lamina in systemic chromosome stretching.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

Cilia-driven asymmetric Hedgehog signalling determines the amphioxus left-right axis by controlling Dand5 expression.

Cilia rotation-driven nodal flow is crucial for the left-right (L-R) break in symmetry in most vertebrates. However, the mechanism by which the flow signal is translated to asymmetric gene expression has been insufficiently addressed. Here, we show that Hedgehog (Hh) signalling is asymmetrically activated (L

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig, Hemimerus talpoides.

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.

Development of pancreatic acini in embryos of the grass snake Natrix natrix (Lepidosauria, Serpentes).

This study report about the differentiation of pancreatic acinar tissue in grass snake, Natrix natrix, embryos using light microscopy, transmission electron microscopy, and immuno-gold labeling. Differentiation of acinar cells in the embryonic pancreas of the grass snake is similar to that of other amniotes. Pancreatic acini occurred for the first time at Stage VIII, which is the midpoint of embryonic development. Two pattern of acinar cell differentiation were observed. The first involved formation of zymogen granules followed by cell migration from ducts. In the second, one zymogen granule was formed at the end of acinar cell differentiation. During embryonic development in the pancreatic acini of N. natrix, five types of zymogen granules were established, which correlated with the degree of their maturation and condensation. Within differentiating acini of the studied species, three types of cells were present: acinar, centroacinar, and endocrine cells. The origin of acinar cells as well as centroacinar cells in the pancreas of the studied species was the pancreatic ducts, which is similar as in other vertebrates. In the differentiating pancreatic acini of N. natrix, intermediate cells were not present. It may be related to the lack of transdifferentiation activity of acinar cells in the studied species. Amylase activity of exocrine pancreas was detected only at the end of embryonic development, which may be related to animal feeding after hatching from external sources that are rich in carbohydrates and presence of digestive enzymes in the egg yolk. Mitotic division of acinar cells was the main mechanism of expansion of acinar tissue during pancreas differentiation in the grass snake embryos.