Weronika Rupik

Embryology of the VNO and associated structures in the grass snake Natrix natrix (Squamata: Natricinae): a 3D perspective

BackgroundSnakes are considered to be vomerolfaction specialists. They are members of one of the most diverse groups of vertebrates, Squamata. The vomeronasal organ and the associated structures (such as the lacrimal duct, choanal groove, lamina transversalis anterior and cupola Jacobsoni) of adult lizards and snakes have received much anatomical, histological, physiological and behavioural attention. However, only limited embryological investigation into these structures, constrained to some anatomical or cellular studies and brief surveys, has been carried out thus far. The purpose of this study was, first, to examine the embryonic development of the vomeronasal organ and the associated structures in the grass snake (Natrix natrix), using three-dimensional reconstructions based on histological studies, and, second, to compare the obtained results with those presented in known publications on other snakes and lizards.ResultsFive major developmental processes were taken into consideration in this study: separation of the vomeronasal organ from the nasal cavity and its specialization, development of the mushroom body, formation of the lacrimal duct, development of the cupola Jacobsoni and its relation to the vomeronasal nerve, and specialization of the sensory epithelium. Our visualizations showed the VNO in relation to the nasal cavity, choanal groove, lacrimal duct and cupola Jacobsoni at different embryonic stages. We confirmed that the choanal groove disappears gradually, which indicates that this structure is absent in adult grass snakes. On our histological sections, we observed a gradual growth in the height of the columns of the vomeronasal sensory epithelium and widening of the spaces between them.ConclusionsThe main ophidian taxa (Scolecophidia, Henophidia and Caenophidia), just like other squamate clades, seem to be evolutionarily conservative at some levels with respect to the VNO and associated structures morphology. Thus, it was possible to homologize certain embryonic levels of the anatomical and histological complexity, observed in the grass snake, with adult conditions of certain groups of Squamata. This may reflect evolutionary shift in Squamata from visually oriented predators to vomerolfaction specialists. Our descriptions offer material useful for future comparative studies of Squamata, both at their anatomical and histological levels.

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.

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake, Natrix natrix L. (Lepidosauria, Serpentes)

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron‐dense crystalline‐like core that was polygonal in shape and an electron‐dense outer zone. The A granules had a spherical electron‐dense eccentrically located core and a moderately electron‐dense outer zone. The D granules were filled with a moderately electron‐dense non‐homogeneous content. The PP granules had a spherical electron‐dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.

Does the grass snake (Natrix natrix) (Squamata: Serpentes: Natricinae) fit the amniotes-specific model of myogenesis?

In the grass snake (Natrix natrix), the newly developed somites form vesicles that are located on both sides of the neural tube. The walls of the vesicles are composed of tightly connected epithelial cells surrounding the cavity (the somitocoel). Also, in the newly formed somites, the Pax3 protein can be observed in the somite wall cells. Subsequently, the somite splits into three compartments: the sclerotome, dermomyotome (with the dorsomedial [DM] and the ventrolateral [VL] lips) and the myotome. At this stage, the Pax3 protein is detected in both the DM and VL lips of the dermomyotome and in the mononucleated cells of the myotome, whereas the Pax7 protein is observed in the medial part of the dermomyotome and in some of the mononucleated cells of the myotome. The mononucleated cells then become elongated and form myotubes. As myogenesis proceeds, the myotome is filled with multinucleated myotubes accompanied by mononucleated, Pax7-positive cells (satellite cells) that are involved in muscle growth. The Pax3-positive progenitor muscle cells are no longer observed. Moreover, we have observed unique features in the differentiation of the muscles in these snakes. Specifically, our studies have revealed the presence of two classes of muscles in the myotomes. The first class is characterised by fast muscle fibres, with myofibrils equally distributed throughout the sarcoplasm. In the second class, composed of slow muscle fibres, the sarcoplasm is filled with lipid droplets. We assume that their storage could play a crucial role during hibernation in the adult snakes. We suggest that the model of myotomal myogenesis in reptiles, birds and mammals shows the same morphological and molecular character. We therefore believe that the grass snake, in spite of the unique features of its myogenesis, fits into the amniotes-specific model of trunk muscle development.