Micromorphological and anatomical investigations on Conringia Heist. ex Fabr.

The micromorphological and anatomical investigations on 6 taxa of Conringia Heist. ex Fabr. growing in Turkey were carried out. The anatomy of root, stem and leaves was examined in detail. In micromorphological studies stem and leaf surface of species was examined. Mesophyll type of lamina is dorsiventral (Conringia planisiliqua, C. persica, C. austriaca) and equifacial (C. clavata, C.grandiflora, C. orientalis). Stomata situated on both surfaces, are anisocytic and rarely anomocytic types. Crystals of calcium oxalate observed in only one species (C. grandiflora) was found to have cubic type parenchyma cells of the stem and leaf. In micromorphological studies the surface of the stem and leaves were found to be glabrous. Epidermal surface of leaves was observed to be covered with waxy cuticular particles. Anticlinal walls of the adaxial cells are undulate and rarely straight and anticlinal walls of abaxial epidermis are undulate and rarely sinuate. Periclinal walls were found to be covered with upper waxy layer which was flat. A diagnostic key based on combined stem, leaf and leaf epidermal characteristics are presented. The leaf mesophyll structures, stem vascular bundles number, lignified or unlignified of sclerenchyma, epidermal surface and absence or presence of crystals were found to be important characters for the identification of Conringia species.

Shh induces symmetry breaking in the presomitic mesoderm by inducing tissue shear and orientated cell rearrangements

Future boundaries of skeletal muscle segments are determined in the presomitic mesoderm (PSM). Within the PSM, future somitic cells undergo significant changes in both morphology and position. How such large-scale cellular changes are coordinated and the effect on the future border formation is unknown. We find that cellular rearrangements differ between cell populations within the PSM. In contrast to lateral somitic cells, which display less organized rearrangement, the adaxial cell layer undergoes significant tissue shearing with dorsal and ventral cells sliding posteriorly. This shear is generated by orientated intercalations of dorsally and ventrally located adaxial cells, which induces a chevron-like pattern. We find Shh signaling is required for the tissue shear and morphogenesis of adaxial cells. In particular, we observe Shh-dependent polarized recruitment of non-muscle myosin IIA drives apical constrictions, and thus the intercalations and shear. This reveals a novel role for Shh in regulating cell mechanics in the PSM.

Floral Nectary Anatomy and Ultrastructure in Mycoheterotrophic Plant,Epipogium aphyllumSw. (Orchidaceae)

Epipogium aphyllumis a European-Asian obligatory mycoheterotrophic orchid containing no chlorophyll. Flowers are not resupinate with a sack-shape spur and cordate lip, which is divided into two parts: the basal (hypochile) and distal one (epichile). The floral analysis provides strong evidence to conclude that nectar is secreted on the upper surface of pink-coloured papillate ridges and epidermal (adaxial) cells at different place in spur, especially at the apex. The exudation on papillae has been observed through the entire anthesis and it has been stained on polysaccharides, proteins, and lipids. The dense cytoplasm of papillae contains profuse endoplasmic reticulum, plentiful vesicles (bigger ones with tannin-like materials), numerous mitochondria, sometimes dictyosomes, starch grains, and plastids with tubular structures. The large electron-dense bodies in cell walls are structurally the same as tannin-like materials from vesicles that are in contact with plasmalemma. The rupture of thin layer of swelled cuticle is caused by pressure of gathered substances exuded due to granulocrine secretion. The idioblasts with raphides occur mainly in tepals tissue. The dynamic changes of the nectar exudation, released through endocrine secretion, have been noticeable during the anthesis: both on the lip and inside the spur. The nectar secretion is not dependent on the colour form ofE. aphyllumblooming shoots. The floral biology and ultrastructure differ from mycoheterotrophic plants known up to date.

Flounder (Paralichthys olivaceus) FoxD1 and its regulation on the expression of myogenic regulatory factor, MyoD

It has been reported that FoxD1 plays important roles in formation of several different tissues, such as retina and kidney in vertebrates. The function of FoxD1 in muscle development is, however, unclear although it is expressed in muscle cells in zebrafish. Muscles are the major tissue in fish, which serves as a rich protein source in our diet. To further understand the function of FoxD1 in fish muscle development, here we isolated and characterized the FoxD1 gene from flounder (Paralichthys olivaceus), a valuable sea food and an important fish species in aquaculture in Asia. We analyzed its expression pattern and function in regulating myogenic regulatory factor, MyoD, one of the earliest marker of myogenic commitment. In situ hybridization revealed that FoxD1 was expressed in the tailbud, adaxial cells, posterior intestine, forebrain, midbrain and half of the retina in flounder embryos. Functional studies demonstrated that when flounder FoxD1 was over-expressed in zebrafish by microinjection, MyoD expression was decreased, suggesting that FoxD1 may be involved in myogenesis by regulating the expression of MyoD.

The Development of Form and Function in Fishes and the Question of Larval Adaptation

<em>Abstract.</em>—Three phases of myogenesis have been identified in the myotomal muscles of larval teleosts. The commitment of embryonic slow and fast muscle lineages is determined prior to segmentation (embryonic myogenesis) and involves notochord and floorplate derived signaling pathways, which drive the adaxial cells to a slow muscle fate. The adaxial cells elongate to span the entire somite width and subsequently migrate through the myotome to form a superficial layer of slow muscle fibers. The remaining cells of the lateral mesoderm adopt the default fast muscle phenotype. The second phase of fiber expansion in the myotomes involves recruitment from discrete germinal zones for both slow and fast muscle fibers (stratified hyperplasia). Finally, myogenic precursor cells are activated throughout the myotome (mosaic hyperplasia). The progeny of these cells either fuse to form additional fibers on the surface of existing muscle fibers or are absorbed by fibers as they expand in diameter (hypertrophic growth). There is considerable species diversity with respect to the timing of innervation of the embryonic muscle fibers in relation to other developmental events, the degree of maturation of the muscle fibers at hatching, and the onset and relative importance of stratified and mosaic hyperplasia to growth during larval life. A subset of myogenic cells specified by their position in the anterior myotomes are thought to migrate out and populate the pectoral fin buds leading to the differentiation of the pectoral fin muscles. Little is known about the mechanism of formation of the unpaired fin muscles, which occurs after the differentiation of the myotomes and is often delayed until relatively late in larval life. During ontogeny, embryonic isoforms of the myofibrillar proteins are replaced by larval and adult isoforms, and the adult multiterminal pattern of slow muscle innervation gradually develops, reflecting changes in swimming style and performance as body size increases. The body length at which particular protein isoforms are switched on varies for each myofibrillar component and with temperature. In general, early larval stages show a greater reliance on aerobic metabolic pathways and a lower capacity for anaerobic glycolysis than later larval and juvenile stages. Temperature has a marked effect on the ultrastructure, number, and phenotype of larval muscle fibers. Recent evidence suggests that egg incubation temperature can influence myogenic cell commitment, producing long-term consequences for fiber recruitment and growth performance during subsequent stages of the life cycle. The ecological significance of the phenotypic plasticity of muscle growth and some potential applications to fisheries science are briefly discussed.

The zebrafish diwanka gene controls an early step of motor growth cone migration

During vertebrate embryogenesis different classes of motor axons exit the spinal cord and migrate on common axonal paths into the periphery. Surprisingly little is known about how this initial migration of spinal motor axons is controlled by external cues. Here, we show that the diwanka gene is required for growth cone migration of three identified subtypes of zebrafish primary motoneurons. In diwanka mutant embryos, motor growth cone migration within the spinal cord is unaffected but it is strongly impaired as motor axons enter their common path to the somites. Chimera analysis shows that diwanka gene activity is required in a small set of myotomal cells, called adaxial cells. We identified a subset of the adaxial cells to be sufficient to rescue the diwanka motor axon defect. Moreover, we show that this subset of adaxial cells delineates the common axonal path prior to axonogenesis, and we show that interactions between these adaxial cells and motor growth cones are likely to be transient. The studies demonstrate that a distinct population of myotomal cells plays a pivotal role in the early migration of zebrafish motor axons and identify the diwanka gene as a somite-derived cue required to establish an axonal path from the spinal cord to the somites.

Mutations affecting somite formation and patterning in the zebrafish, Danio rerio

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

Mutations affecting the formation of the notochord in the zebrafish, Danio rerio

In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes, floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.

Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation

We have examined the development of specific muscle fiber types in zebrafish axial muscle by labeling myogenic precursor cells with vital fluorescent dyes and following their subsequent differentiation and fate. Two populations of muscle precursors, medial and lateral, can be distinguished in the segmental plate by position, morphology and gene expression. The medial cells, known as adaxial cells, are large, cuboidal cells adjacent to the notochord that express myoD. Surprisingly, after somite formation, they migrate radially away from the notochord, becoming a superficial layer of muscle cells. A subset of adaxial cells develop into engrailed-expressing muscle pioneers. Adaxial cells differentiate into slow muscle fibers of the adult fish. We have named the lateral population of cells in the segmental plate, lateral presomitic cells. They are smaller, more irregularly shaped and separated from the notochord by adaxial cells; they do not express myoD until after somite formation. Lateral presomitic cells remain deep in the myotome and they differentiate into fast muscle fibers. Thus, slow and fast muscle fiber types in zebrafish axial muscle arise from distinct populations of cells in the segmental plate that develop in different cellular environments and display distinct behaviors.