Ultrastructural features of the differentiating thyroid primordium in the sand lizard (Lacerta agilis L.) from the differentiation of the cellular cords to the formation of the follicular lumen.

The differentiation of the thyroid primordium of lacertilian species is poorly understood. The present study reports on the ultrastructural analysis of the developing thyroid primordium in the sand lizard (Lacerta agilis) during the early stages of differentiation. The early thyroid primordium of sand lizard embryos was composed of cellular cords that contained single cells with a giant lipid droplet, which were eliminated by specific autophagy (lipophagy). The follicular lumens at the periphery of the primordium differentiated even before the division of the cellular cords. When the single cells within the cords started to die through paraptosis, the adjacent cells started to polarise and junctional complexes began to form around them. After polarisation and clearing up after the formation of the lumens, the cellular cords divided into definitive follicles. The cellular cords in the central part of the primordium started to differentiate later than those at the periphery. The cellular cords divided into presumptive follicles first and only later differentiated into definitive follicles. During this process, a population of centrally located cells was removed through apoptosis to form the lumen. Although the follicular lumen in sand lizard embryos is differentiated by cavitation similar to that in the grass snake, there were very important differences during the early stages of the differentiation of the cellular cords and the formation of the thyroid follicles.

Unique features of myogenesis in Egyptian cobra (Naja haje) (Squamata: Serpentes: Elapidae).

During early stages of myotomal myogenesis, the myotome of Egyptian cobra (Naja haje) is composed of homogenous populations of mononucleated primary myotubes. At later developmental phase, primary myotubes are accompanied by closely adhering mononucleated cells. Based on localization and morphology, we assume that mononucleated cells share features with satellite cells involved in muscle growth. An indirect morphological evidence of the fusion of mononucleated cells with myotubes is the presence of numerous vesicles in the subsarcolemmal region of myotubes adjacent to mononucleated cell. As differentiation proceeded, secondary muscle fibres appeared with considerably smaller diameter as compared to primary muscle fibre. Studies on N. haje myotomal myogenesis revealed some unique features of muscle differentiation. TEM analysis showed in the N. haje myotomes two classes of muscle fibres. The first class was characterized by typical for fast muscle fibres regular distribution of myofibrils which fill the whole volume of muscle fibre sarcoplasm. White muscle fibres in studied species were a prominent group of muscles in the myotome. The second class showed tightly paced myofibrils surrounding the centrally located nucleus accompanied by numerous vesicles of different diameter. The sarcoplasm of these cells was characterized by numerous lipid droplets. Based on morphological features, we believe that muscle capable of lipid storage belong to slow muscle fibres and the presence of lipid droplets in the sarcoplasm of these muscles during myogenesis might be a crucial adaptive mechanisms for subsequent hibernation in adults. This phenomenon was, for the first time, described in studies on N. haje myogenesis.

Reptilian myotomal myogenesis-lessons from the sand lizard Lacerta agilis L. (Reptilia, Lacertidae)Update.

Reptilian myotomal myogenesis is poorly understood. This paper reports on structural, ultrastructural and immunocytochemical studies of muscle differentiation in sand lizard (Lacerta agilis) embryos. During somitogenesis, the somites are composed of epithelial vesicles with a centrally located somitocoel. At later developmental stages the ventral portion of the somite cortex disaggregates into the sclerotome mesenchyme, while the dorsal wall of the somite differentiates into dermomyotome. At these developmental stages, mononucleated cells of the dermomyotome are Pax3-positive. The dermomyotome layer forms the dorsomedial and ventromedial lips. The myotome is first composed of mono- and then of multinucleated myotubes and small mononucleated cells that occur in the vicinity of the myotubes. These mononucleated cells exhibit low proliferative potential as revealed by the use of PCNA antibody. At subsequent stages of myogenesis the mononucleated cells express Pax7 protein, a marker of satellite cells, and assume ultrastructural features characteristic of satellite cells. Some of the mononucleated cells contribute to muscle growth, being involved in fusion with differentiating muscle fibers. This study revealed similarities of myotomal myogenesis in reptiles to that of other vertebrates.

Cross-immunoreactivity between the LH1 antibody and cytokeratin epitopes in the differentiating epidermis of embryos of the grass snake Natrix natrix L. during the end stages of embryogenesis.

The monoclonal anti-cytokeratin 1/10 (LH1) antibody recognizing K1/K10 keratin epitopes that characterizes a keratinized epidermis of mammals cross-reacts with the beta and Oberhäutchen layers covering the scales and gastrosteges of grass snake embryos during the final period of epidermis differentiation. The immunolocalization of the anti-cytokeratin 1/10 (LH1) antibody appears in the beta layer of the epidermis, covering the outer surface of the gastrosteges at the beginning of developmental stage XI, and in the beta layer of the epidermis, covering the outer surface of the scales at the end of developmental stage XI. This antibody cross-reacts with the Oberhäutchen layers in the epidermis covering the outer surface of both scales and gastrosteges at developmental stages XI and XII just before its fusion with the beta layers. After fusion of the Oberhäutchen and beta layers, LH1 immunolabeling is weaker than before. This might suggest that alpha-keratins in these layers of the epidermis are masked by beta-keratins, modified, or degraded. The anti-cytokeratin 1/10 (LH1) antibody stains the Oberhäutchen layer in the epidermis covering the inner surface of the gastrosteges and the hinge regions between gastrosteges at the end of developmental stage XI. However, the Oberhäutchen of the epidermis covering the inner surfaces of the scales and the hinge regions between scales does not show cytokeratin 1/10 (LH1) immunolabeling until hatching. This cross-reactivity suggests that the beta and Oberhäutchen layers probably contain some alpha-keratins that react with the LH1 antibody. It is possible that these alpha-keratins create specific scaffolding for the latest beta-keratin deposition. It is also possible that the LH1 antibody cross-reacts with other epidermal proteins such as filament-associated proteins, i.e., filaggrin-like. The anti-cytokeratin 1/10 (LH1) antibody does not stain the alpha and mesos layers until hatching. We suppose that the differentiation of these layers will begin just after the first postnatal sloughing.

Ultrastructural studies of epidermis keratinization in grass snake embryos Natrix natrix L. (Lepidosauria, Serpentes) during late embryogenesis.

The changes and biochemical features of the epidermis that accompany the differentiation and embryonic shedding complex formation in grass snake Natrix natrix L. embryos were studied ultrastructurally and immunocytochemically with two panels of antibodies (AE1, AE3, AE1/AE3; anti-cytokeratin, pan mixture, Lu-5 and PCK-26). All observed changes in the ultrastructure of the cells forming the epidermal layers were associated with the physiological changes that occurred in the embryonic epidermis, such as changing of the manner of nutrition and keratinization leading to the embryonic shedding complex formation. The layers that originated first (basal, outer and inner periderm and clear layer) differentiated very early and rapidly. Rapid differentiation was also observed in the layers that are very important for the functioning of the epidermis in Natrix embryos (oberhäutchen and beta-layers). They started to differentiate at developmental stage IX, and then fused and formed the embryonic shedding complex at developmental stage XI. During the embryonic development of the grass snake the smallest changes appeared in the ultrastructure of the cells in the mesos and alpha-layers because they perform supplementary functions in the process of embryonic molting. They were undifferentiated until the end of embryonic development and started to differentiate just before the first adult molting. AE1/AE3, anti-cytokeratin, pan mixture, Lu-5 and PCK-26 antibodies immunolabeled clear layer, oberhäutchen and beta-layers at the latest phase of developmental stage XI. It should be noted that these antibodies did not immunolabel the alpha-layer until hatching. The presence of alpha-keratin immunolabeling in layers that were keratinized, particularly in the oberhäutchen and beta-layers in embryos, indicated that they were not as hard as in fully mature individuals.