Intestinal coelomic transplants: a novel method for studying enteric nervous system development

Normal development of the enteric nervous system (ENS) requires the coordinated activity of multiple proteins to regulate the migration, proliferation, and differentiation of enteric neural crest cells. Much of our current knowledge of the molecular regulation of ENS development has been gained from transgenic mouse models and cultured neural crest cells. We have developed a method for studying the molecular basis of ENS formation complementing these techniques. Aneural quail or mouse hindgut, isolated prior to the arrival of neural crest cells, was transplanted into the coelomic cavity of a host chick embryo. Neural crest cells from the chick host migrated to and colonized the grafted hindgut. Thorough characterization of the resulting intestinal chimeras was performed by using immunohistochemistry and vital dye labeling to determine the origin of the host-derived cells, their pattern of migration, and their capacity to differentiate. The formation of the ENS in the intestinal chimeras was found to recapitulate many aspects of normal ENS development. The host-derived cells arose from the vagal neural crest and populated the graft in a rostral-to-caudal wave of migration, with the submucosal plexus being colonized first. These crest-derived cells differentiated into neurons and glial cells, forming ganglionated plexuses grossly indistinguishable from normal ENS. The resulting plexuses were specific to the grafted hindgut, with quail grafts developing two ganglionated plexuses, but mouse grafts developing only a single myenteric plexus. We discuss the advantages of intestinal coelomic transplants for studying ENS development. This work was supported by NIH K08HD46655 (to A.M.G.).     

Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system

Neural crest cells (NCC) migrate, proliferate, and differentiate within the wall of the gastrointestinal tract to give rise to the neurons and glial cells of the enteric nervous system (ENS). The intestinal microenvironment is critical in this process and endothelin-3 (ET3) is known to have an essential role. Mutations of this gene cause distal intestinal aganglionosis in rodents, but its mechanism of action is poorly understood. We find that inhibition of ET3 signaling in cultured avian intestine also leads to hindgut aganglionosis. The aim of this study was to determine the role of ET3 during formation of the avian hindgut ENS. To answer this question, we created chick-quail intestinal chimeras by transplanting preganglionic quail hindguts into the coelomic cavity of chick embryos. The quail grafts develop two ganglionated plexuses of differentiated neurons and glial cells originating entirely from the host neural crest. The presence of excess ET3 in the grafts results in a significant increase in ganglion cell number, while inhibition of endothelin receptor-B (EDNRB) leads to severe hypoganglionosis. The ET3-induced hyperganglionosis is associated with an increase in enteric crest cell proliferation. Using hindgut explants cultured in collagen gel, we find that ET3 also inhibits neuronal differentiation in the ENS. Finally, ET3, which is strongly expressed in the ceca, inhibits the chemoattraction of NCC to glial-derived neurotrophic factor (GDNF). Our results demonstrate multiple roles for ET3 signaling during ENS development in the avian hindgut, where it influences NCC proliferation, differentiation, and migration.     

Elektronenmikroskopische Untersuchungen an der Froschlunge

Zusammenfassung Das Alveolarepithel der Froschlunge weist nur einen einzigen Zelltyp auf. Die Zellkörper sitzen in den Nischen zwischen den Kapillaren, die sie mit Zytoplasmaausläufern überdecken. Die Epithelzellen enthalten große Zytosomen mit osmiophilen Lamellen mit einer Periode von 40–42 Å. Sie sind den Typ II-Pneumozyten der Säugerlunge vergleichbar.Das Alveolarepithel der Froschlunge ist mit einer Grenzschicht bedeckt, die in Abhängigkeit von der Fixierung eine 40–42 Å-Periode aufweist oder aus einer oder mehreren Doppelmembranen zusammengesetzt ist. Gittermuster und Myelinfiguren sind vorhanden. Das bedeutet, daß Surfactant in der Froschlunge in gleicher Weise wie in der Säugerlunge dargestellt werden kann.

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Electron microscopic studies on the lung of the frogI. Demonstration of the alveolar lining layer (surfactant)

Summary The alveolar epithelium of the frog exhibits only one type of cells. The cell-bodies are situated in the spaces among the capillaries, which they cover with cytoplasmic extensions. The epithelial cells contain large bodies (cytosomes) with osmiophilic lamellae having a period of 40–42 Å. The alveolar cells are considered to be similar to the type II-pneumocytes of mammalian lungs.The alveolar epithelium of the lung of the frog is covered with a lining layer, which depending on the method of fixation consists of periods of 40–42 Å or of one or more double membranes. Lattice formations and myelin figures are seen. This means that the surfactant in the lung of the frog can be demonstrated in the same way as in mammalian lungs.

     

TNAP, TrAP, ecto-purinergic signaling, and bone remodeling

Bone remodeling is a process of continuous resorption and formation/mineralization carried out by osteoclasts and osteoblasts, which, along with osteocytes, comprise the bone multicellular unit (BMU). A key component of the BMU is the bone remodeling compartment (BRC), isolated from the marrow by a canopy of osteoblast-like lining cells. Although much progress has been made regarding the cytokine-dependent and hormonal regulation of bone remodeling, less attention has been placed on the role of extracellular pH (pH(e)). Osteoclastic bone resorption occurs at acidic pH(e). Furthermore, osteoclasts can be regarded as epithelial-like cells, due to their polarized structure and ability to form a seal against bone, isolating the lacunar space. The major ecto-phosphatases of osteoclasts and osteoblasts, acid and alkaline phosphatases, both have ATPase activity with pH optima several units different from neutrality. Furthermore, osteoclasts and osteoblasts express plasma membrane purinergic P2 receptors that, upon activation by ATP, accelerate bone osteoclast resorption and impair osteoblast mineralization. We hypothesize that these ecto-phosphatases help regulate [ATP](e) and localized pH(e) at the sites of bone resorption and mineralization by pH-dependent ATP hydrolysis coupled with P2Y-dependent regulation of osteoclast and osteoblast function. Furthermore, osteoclast cellular HCO3(-), formed as a product of lacunar V-ATPase H(+) secretion, is secreted into the BRC, which could elevate BRC pH(e), in turn affecting osteoblast function. We will review the existing data addressing regulation of BRC pH(e), present a hypothesis regarding its regulation, and discuss the hypothesis in the context of the function of proteins that regulate pH(e).     

Nucleotide pool changes in coelomic cells (eleocytes) of the polychaete Nereis virens during sexual maturation

Eleocytes (a type of coelomic cell) of the polychaete Nereis virens can store large amounts of adenine nucleotides at certain times. Since eleocytes have specific functions related to gametogenesis, we tested whether the presence of these large nucleotide stores in eleocytes is specific to gender or related to specific events during gametogenesis. Nucleotide pools in eleocytes isolated at different stages of sexual maturation from N. virens were analysed using high-performance liquid chromatography. Eleocytes from immature and male animals had extremely high concentrations of both AMP and ADP (each > 10 μmol/ml of packed cell volume). In eleocytes from male animals, the high nucleotide stores were maintained throughout the maturation phase and decreased at a late stage, while in female animals the nucleotides were degraded at an early stage of maturation. In male eleocytes, the decrease in the adenine nucleotide pool may be the result of its conversion to inosine which is then released by the eleocytes and reutilized by male germ cells for nucleic acid biosynthesis, as has been suggested previously. Our study shows that the time of degradation of the adenine nucleotide pool coincides with the period of spermatogonia proliferation which involves intense nucleic acid synthesis. ATP levels (0.4–1.5 μmol/ml packed cell volume) and the guanine nucleotide pool (GTP+GDP+GMP; 0.08–0.18 μmol/ml packed cell volume) were similar in both sexes, did not change during germ cell development and were decreased only in eleocytes from prespawning females. The GTP/GDP ratios were initially higher (up to 14) in eleocytes from females compared to ratios in eleocytes from immature (4–9) and male animals (up to 8), and decreased during the maturation phase of the animals. GTP levels were correlated with those of ATP; this correlation was much closer in eleocytes from females than from males. The results further support the hypothesis that the adenine nucleotide stores in the eleocytes are maintained as a supply of purine precursors for the growing germ cells.     

Ultrastructure of the coelom in the brachiopod Lingula anatina

The organization of the coelomic system and the ultrastructure of the coelomic lining are used in phylogenetic analysis to establish the relationships between major taxa. Investigation of the anatomy and ultrastructure of the coelomic system in brachiopods, which are poorly studied, can provide answers to fundamental questions about the evolution of the coelom in coelomic bilaterians. In the current study, the organization of the coelom of the lophophore in the brachiopod Lingula anatina was investigated using semithin sectioning, 3D reconstruction, and transmission electron microscopy. The lophophore of L. anatina contains two main compartments: the preoral coelom and the lophophoral coelom. The lining of the preoral coelom consists of ciliated cells. The lophophoral coelom is subdivided into paired coelomic sacs: the large and small sinuses (= canals). The lining of the lophophoral coelom varies in structure and includes monociliate myoepithelium, alternating epithelial and myoepithelial cells, specialized peritoneum and muscle cells, and podocyte‐like cells. Connections between cells of the coelomic lining are provided by adherens junctions, tight‐like junctions, septate junctions, adhesive junctions, and direct cytoplasmic bridges. The structure of the coelomic lining varies greatly in both of the main stems of the Bilateria, that is, in the Protostomia and Deuterostomia. Because of this great variety, the structure of the coelomic lining cannot by itself be used in phylogenetic analysis. At the same time, the ciliated myoepithelium can be considered as the ancestral type of coelomic lining. The many different kinds of junctions between cells of the coelomic lining may help coordinate the functioning of epithelial cells and muscle cells.     

Colonisation of soft lining materials by micro‐organisms

doi:10.1111/j.1741‐2358.2009.00300.x
Colonisation of soft lining materials by micro‐organisms Objective: This study evaluated the in vitro adherence of pathogenic micro‐organisms, Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa, to soft lining materials and their inhibitory effect on these micro‐organisms. Materials and Methods: To measure adherence, specimens of Molloplast B and Ufi Gel P were inoculated [10⁷ colony‐forming units per millimetre (cfu/ml)] with TSB media containing the micro‐organisms. To determine the number of micro‐organisms in the 10^?2–10^?5 dilutions, 25 μl of the suspension were transferred to plates of selective media. Colony counts of each specimen were quantified (cfu/ml). The surface roughness was measured with a perfilometer to assess the relationship between the adherence of micro‐organisms and surface roughness of each material. For the inhibition test, specimens of materials were placed in agar plates inoculated individually with the micro‐organisms. After 48 h, the inhibition zones around the specimens were measured. Results: None of the materials exhibited inhibition zones. The number of cfu/ml of S. aureus and P. aeruginosa were significantly greater than C. albicans for both materials. The Ufi Gel P exhibited greater adherence of C. albicans than Molloplast B. No correlation was observed between the adherence of micro‐organisms and surface roughness. Conclusion: The surface roughness of the materials is not the only factor governing micro‐organism adherence.     

Parathyroid hormone (PTH) and hematopoiesis: new support for some old observations

Forty-seven years ago, the parathyroid hormone (PTH) in one injection of Lilly's old bovine parathyroid extract, PTE, was found to greatly increase the 30-day survival of heavily X-irradiated rats when given from 18 h before to as long as 3 h after irradiation but no later. This was the first indication that PTH might stimulate hematopoiesis. Recent studies have confirmed the relation between PTH and hematopoiesis by showing that hPTH-(1-34)OH increases the size of the hematopoietic stem cell pool in mice. The peptide operates through a cyclic AMP-mediated burst of Jagged 1 production in osteoblastic cells lining the stem cells' niches on trabecular bone surfaces. The osteoblastic cells' Jagged 1 increases the hematopoietic stem cell pool by activating Notch receptors on attached stem cells. PTH-triggered cyclic AMP signals also directly stimulate the proliferation of the hematopoietic stem cells. However, the single PTH injection in the early experiments using PTE probably increased the survival of irradiated rats mainly by preventing the damaged hematopoietic progenitors from irreversibly initiating self-destructive apoptogenesis during the first 5 h after irradiation. It has also been shown that several daily injections of hPTH-(1-34)OH enable lethally irradiated mice to survive by stimulating the growth of transplanted normal bone marrow cells. If the osteogenic PTHs currently entering or on the verge of entering the market for treating osteoporosis can also drive hematopoiesis in humans as well as rodents, they could be potent tools for reducing the damage inflicted on bone marrow by cytotoxic cancer chemotherapeutic drugs and ionizing radiation.