Systematic analysis of hematopoietic, vasculogenetic, and angiogenetic phases in the developing embryonic avian lung, Gallus gallus variant domesticus

In the embryonic lung of the domestic fowl, Gallus gallus variant domesticus, hematogenetic and vasculogenetic cells become ultrastructurally clear from day 4 of development. In the former group of cells, filopodial extensions coalesce, cytoplasm thickens, and accumulating hemoglobin displaces the nucleus peripherally while in the latter, conspicuous filopodial extensions and large nuclei develop as the cells assume a rather stellate appearance. From day 5, erythrocytes and granular leukocytes begin forming from cytoarchitecturally cognate hematogenetic cells. The cells become distinguishable when hemoglobin starts to accumulate in the erythroblasts and electron dense bodies form in the leukoblasts. Vasculogenesis begins from day 7 in different areas of the developing lung: erthrocytes (but not granular leukocytes) appear to attract committed vasculogenetic cells (angioblasts) that form an endothelial lining and vessel wall. Arrangement of angioblasts around forming blood vessels sets the direction along which the vessels sprout (angiogenesis). In some areas of the developing lung, through what seems like an inductive erythropoietic process, arcades of erythrocytes organize. Once endothelial cells surround such continuities, discrete vascular units organize. By day 10, the major parts of the in-built (intrinsic) pulmonary vasculature are assembled. Complete pulmonary circulation (i.e., through the exchange tissue) is not established until after day 18 when the blood capillaries start to develop. Since the precursory erythrocytes do not have a respiratory role, it is imperative that de novo erythropoiesis is essential for vasculogenesis. Diffuse (fragmentary) development and subsequent piecemeal assembly of the pulmonary vascular system may explicate the fabrication of a complex circulatory architecture that grants cross-current, counter-current, and multicapillary serial arterialization designs in the exchange tissue of the avian lung. The exceptional respiratory efficiency of the avian lung is largely attributable to the geometries (physical interfacing) between the bronchial and vascular elements at different levels of morphological organization.     

Novel lipophilic amidate oxorhenium and oxotechnetium complexes as potential brain agents: synthesis, characterization and biological evaluation

Novel oxorhenium and oxotechnetium complexes based on the tetradentate 1-(2-hydroxybenzamido)-2-(pyridinecarboxamido)benzene, H3L, ligand have been synthesized and characterized herein. Thus, by reacting equimolar quantities of the triply deprotonated ligand L3- with the suitable MO3+ precursor, the following neutral MOL complexes could be easily produced following similar synthetic routes: M = Re (1), M = 99gTc (2), and M = 99mTc (3). Complexes 1 and 2, prepared in macroscopic amounts, were chemically characterized and their structure determined by single-crystal X-ray analysis. They are isostructural metal chelates, adopting a distorted square pyramidal geometry around the metal. The N3O donor atom set of the tetradentate ligand defines the basal plane and the oxygen atom of the M = O core occupies the apex of the pyramid. Complex 3 forms quantitatively at tracer level by mixing the H3L ligand with Na99mTcO4 generator eluate in aqueous alkaline media and using tin chloride as reductant in the presence of citrate. Its structure was established by chromatographic comparison with prototypic complexes 1 and 2 using high-performance liquid chromatographic techniques. When challenged with excess glutathione in vitro, complex 3 is rapidly converted to hydrophilic unidentified metal species. Tissue distribution data after administration of complex 3 in vivo revealed a significant uptake and retention of this compound in brain tissue.     

Functional morphology of the gas-gland cells of the air-bladder of Oreochromis alcalicus grahami (teleostei: cichlidae): an ultrastructural study on a fish adapted to a severe, highly alkaline environment

Oreochromis alcalicus grahami is a small cichlid fish that lives in shallow peripheral lagoons of Lake Magadi, Kenya. The internal surface of the air-bladder is highly vascularized, illustrating possible utilization as an accessory respiratory organ. The wall of the bladder consists of five distinct tissue layers. From the outer to the inner surfaces are: a squamous, undifferentiated epithelial cell; a collagen-elastic tissue space; a smooth muscle tissue band; an inner connective tissue space; and columnar gas-gland cells projecting into the lumen. The cell membrane over the perikarya of the gas-gland cells was sporadically broken. The disruptions were ascribed to possible physical damage by discharging gas-bubbles. Juxtaluminally, the gas-gland cells attached across tight junctions. The cells have highly euchromatic nuclei and conspicuously large intracytoplasmic secretory bodies. On the blood capillary facing (basal) aspect, the cell membrane of the gas-gland cells is highly amplified. The cells insert onto a smooth muscle tissue band. The morphological features and the topographical arrangement of the gas-gland cells in O. a. grahami are indicative of an operative exchange of materials between them and the underlying tissue components especially the blood capillaries. For a fish that subsists in hot, highly saline and alkaline water heavily invested by avian predators and where the partial pressure of oxygen diurnally shifts from virtual anoxia to hyperoxia, development of a versatile air-bladder for efficient buoyancy control conforms to the functional demands placed on it by a unique environment. Illustratively, instead of the gas-gland morphology in O. a. grahami resembling that in the freshwater fishes, the group from which the fish has evolved, it compares more closely to that of the marine fish. This similarity suggests amazing parallel evolutionary adaptation to biophysically corresponding aquatic milieus.     

Locations, ultrastructural morphology, and putative functions of the branchial podocytes of the fresh water crab Potamon niloticus - Savigy (crustacea, decapoda, potamonidae)

The gills of the African fresh water crab, Potamon niloticus, were investigated by transmission electron microscopy to verify the presence, outline the location(s) of, and describe the ultrastructural attributes of the branchial podocytes (BPs). Topographically, the cells were diffusely distributed in the gills. They were found at the arterial ends of the intralamellar spaces (ILSs), in the efferent hemolymphatic vessel (EHV), in the gill shaft, and in the marginal and central ILSs. In the EHV and the terminal ends of the ILSs, the BPs occurred in cohesive clusters of from three to eight large cells which were affixed to the vessel wall by small fibrocytic cells and bands of myofibrils. In the clusters, the BPs attached directly across interspersed junctional complexes (separated by wide intercellular spaces) and indirectly over a common basement membrane. Abundant heteromorphic, variably electron-dense vacuoles were scattered in the cytoplasm, apparently displacing the nuclei peripherally. The plasmalemma of the BPs were amplified into feet processes (pedicels) which inserted onto a basement membrane. The feet were joined by a thin unit membrane (diaphragm), leaving subcisternal spaces which contained flocculent to granular electron-dense material. The general ultrastructural morphology of the BPs of Potamon was similar to that of other crustaceans. However, atypically, a labyrinth of intercellular spaces (reckoned to be filtratory channels) was observed in the cell clusters. An ultrafiltration role was attested by the characteristic specializations of the plasmalemma and a phagocytic one inferred from the conspicuous intracytoplasmic vacuolation, presence of phagosomes, and overt necrosis and desquamation of the outlying cells of the clusters. The topographic location of the BPs in the EHV and at the terminal ends of the ILSs was perceived to be a strategic arrangement for promoting the detoxification and destruction of harmful materials and invasive agents which pass through the gills, an organ that presents an extensive surface area that interfaces with the ambient medium.     

Male Ornamentation in a Sex‐Role Reversed Pipefish Corythoichthys haematopterus

Sexual selection theory predicts that mating competition in sex‐role reversed animals acts more strongly on females than males and consequently females are expected to develop secondary sexual traits. However, in a sex‐role reversed pipefish Corythoichthys haematopterus (family: Syngnathidae), only males develop an ornamental trait on the thorax, consisting of approx. 3–5 speckles alternated by lateral stripes of brilliant light blue and orange. To understand the function of this male ornament, we examined whether the presence of females affects the expression of this trait, and whether the expression of this trait depends on the male’s physical condition. Individual males were reared in a tank for a month in four different conditions: in high or low food supply and in the presence or absence of a female. After 1 mo, males in better condition expressed larger and deeper blue and yellow speckles, and males maintained with a female expressed larger and deeper blue speckles than solely reared males. These results indicated that the male ornament functions as a signal conveying information on the phenotypic quality of its holder and that females are potential receivers of this signal. Because C. haematopterus exhibits strict monogamy and competition for a mate occurs only among females, we concluded that the male ornament is not displayed in the context of mating competition but rather it is used as a cue for partner recognition to maintain pair bond.     

Complement dependent amplification of the innate response to a cognate microbial ligand by the long pentraxin PTX3

The long pentraxin PTX3 is a fluid-phase pattern recognition receptor, which plays a nonredundant role in resistance against selected pathogens. PTX3 has properties similar to Abs; its production is induced by pathogen recognition, it recognizes microbial moieties, activates complement, and facilitates cellular recognition by phagocytes. The mechanisms responsible for the effector function of PTX3 in vivo have not been elucidated. OmpA, a major outer membrane protein of Gram-negative Enterobacteriaceae, is a microbial moiety recognized by PTX3. In the air pouch model, KpOmpA induces an inflammatory response, which is amplified by coadministration of PTX3 in terms of leukocyte recruitment and proinflammatory cytokine production. PTX3 did not affect the inflammatory response to LPS, a microbial moiety not recognized by PTX3. As PTX3 binds to C1q and modulates the activation of the complement cascade, we assessed the involvement of complement in the amplification of the response elicited by KpOmpA and PTX3. Experiments performed using cobra venom factor, C1-esterase inhibitor, and soluble complement receptor 1 indicate that PTX3 amplifies the inflammatory response to KpOmpA through complement activation. The results reported here demonstrate that PTX3 activates a complement-dependent humoral amplification loop of the innate response to a microbial ligand.     

Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.     

Coupling Met to specific pathways results in distinct developmental outcomes.

Receptor tyrosine kinases (RTKs) mediate distinct biological responses by stimulating similar intracellular signaling pathways. Whether the specificity of the response is determined by qualitative or quantitative differences in signaling output is not known. We addressed this question in vivo by replacing the multifunctional docking sites of Met, the receptor for hepatocyte growth factor, with specific binding motifs for phosphatidylinositol-3 kinase, Src tyrosine kinase, or Grb2 (Met(2P), Met(2S), and Met(2G), respectively). All three mutants retained normal signaling through the multiadaptor Gab1, but differentially recruited specific effectors. While Met(2G) mice developed normally, Met(2P) and Met(2S) mice were loss-of-function mutants displaying different phenotypes and rescue of distinct tissues. These data indicate that RTK-mediated activation of specific signaling pathways is required to fulfill cell-specific functions in vivo.     

A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells

The cloaca serves as a common opening to the urinary and digestive systems. In most mammals, the cloaca is present only during embryogenesis, after which it undergoes a series of septation events leading to the formation of the anal canal and parts of the urogenital tract. During embryogenesis it is surrounded by skeletal muscle. The origin and the mechanisms regulating the development of these muscles have never been determined. Here, we show that the cloacal muscles of the chick originate from somites 30-34, which overlap the domain that gives rise to leg muscles (somites 26-33). Using molecular and cell labelling protocols, we have determined the aetiology of cloacal muscles. Surprisingly, we found that chick cloacal myoblasts first migrate into the developing leg bud and then extend out of the ventral muscle mass towards the cloacal tubercle. The development of homologous cloacal/perineal muscles was also examined in the mouse. Concordant with the results in birds, we found that perineal muscles in mammals also develop from the ventral muscle mass of the hindlimb. We provide genetic evidence that the perineal muscles are migratory, like limb muscles, by showing that they are absent in metd/d mutants. Using experimental embryological procedures (in chick) and genetic models (in chick and mouse), we show that the development of the cloacal musculature is dependent on proximal leg field formation. Thus, we have discovered a novel developmental mechanism in vertebrates whereby muscle cells first migrate from axially located somites to the pelvic limb, then extend towards the midline and only then differentiate into the single cloacal/perineal muscles.