The HOX-like gene Cnox2-Pc is expressed at the anterior region in all life cycle stages of the jellyfish Podocoryne carnea.

The marine jellyfish Podocoryne carnea (Cnidaria, Hydrozoa) has a metagenic life cycle consisting of a larva, a colonial polyp and a free-swimming jellyfish (medusa). To study the function of HOX genes in primitive diploblastic animals we screened a library of P. carnea cDNA using PCR primers derived from the most conserved regions in helix 1 and helix 3 of the homeobox. A novel gene, Cnox2-Pc, has been isolated and characterized. Cnox2-Pc is a HOX cluster-like gene, and its homeodomain shows similarity to the Deformed subfamily of HOM-C/HOX genes. In situ hybridization revealed that Cnox2-Pc is expressed in the anterior region of the larva, the polyp head, and the most apical ectoderm of the differentiating bud during medusa development. In adult medusa expression is restricted to the gastrovascular entoderm. The results suggest that Cnox2-Pc is involved in establishment of an anterior-posterior axis during development in primitive metazoans.

Substrate-dependent interactions of leech microglial cells and neurons in culture.

The principal aim of the present experiments has been to analyze the properties of microglial cells and their role in nerve regeneration. In the leech, damage to the CNS has been shown to be followed by accumulation of laminin and microglial cells at the site of injury (Masuda-Nakagawa et al., 1990. Proc. R. Soc. Lond. B. 241:201-206; and 1993. Proc. Natl. Acad. Sci. USA 90:4966-4970). Procedures were devised for isolating these small, wandering cells from the CNS of the leech. In culture, they were reliably identified by their sizes, shapes, and phagocytotic activity. Their morphology, motility, and interactions with neurons were influenced by the substrate molecules on which they were plated. On the plant lectin concanavalin A (Con A) microglia had a rounded shape and remained stationary. By contrast on extracts of leech extracellular matrix (ECM) enriched with laminin the cells were mobile and spindle-shaped with long processes. On Con A, neuronal growth cones avoided microglial cells, whereas on ECM extract the presence of a microglial cell did not influence neurite growth. Microglial cells showed immunoreactivity on both substrates when stained with a monoclonal antibody against leech laminin. Together these results suggest that microglial cells are influenced in their properties by molecules in the environment and that they could contribute to neuronal outgrowth at the site of an injury.

Axonal sprouting and laminin appearance after destruction of glial sheaths.

Laminin, a large extracellular matrix molecule, is associated with axonal outgrowth during development and regeneration of the nervous system in a variety of animals. In the leech central nervous system, laminin immunoreactivity appears after axon injury in advance of the regenerating axons. Although studies of vertebrate nervous system in culture have implicated glial and Schwann cells as possible sources, the cells that deposit laminin at sites crucial for regeneration in the living animal are not known. We have made a direct test to determine whether, in the central nervous system of the leech, cells other than ensheathing glial cells can produce laminin. Ensheathing glial cells of adult leeches were ablated selectively by intracellular injection of a protease. As a result, leech laminin accumulated within 10 days in regions of the central nervous system where it is not normally found, and undamaged, intact axons began to sprout extensively. In normal leeches laminin immunoreactivity is situated only in the basement membrane that surrounds the central nervous system, whereas after ablation of ensheathing glia it appeared in spaces through which neurons grew. Within days of ablation of the glial cell, small mobile phagocytes, or microglia, accumulated in the spaces formerly occupied by the glial cell. Microglia were concentrated at precisely the sites of new laminin appearance and axon sprouting. These results suggest that in the animal, as in culture, leech laminin promotes sprouting and that microglia may be responsible for its appearance.

The role of matrix molecules in regeneration of leech CNS.

Extracellular matrix (ECM) molecules extracted from the leech central nervous system (CNS) provide substrates that induce extensive growth of processes of identified leech nerve cells in culture. Two ECM molecules, laminin and tenascin, have been identified. The laminin-like molecule has been purified and shown to be a cross-shaped molecule similar to vertebrate laminin with subunits of 340, 220, 180, and 160 kD. Purified laminin as a substrate induces rapid outgrowth of Retzius (R) and Anterior Pagoda (AP) cells in culture. The tenascin molecule has been partially purified. In electronmicrographs, leech tenascin, like vertebrate tenascin, has six arms of equal size joined in a central globule. Highly enriched fractions of leech tenascin induce rapid and extensive outgrowth of Retzius and AP cells in culture. Substrate molecules not only induce outgrowth of processes but also affect the growth patterns of individual nerve cells. Neurites are straight with few branches in laminin, but curved with profuse branches on tenascin. During regeneration of the CNS in the animal, laminin appears at new sites associated with growth cones. The appearance of laminin correlates with the accumulation of microglial cells. Thus, ECM molecules with growth-promoting activity for leech nerve cells in vitro appear to be involved in inducing regeneration and allowing the neurites to reconnect with former targets.

Extracellular matrix molecules in development and regeneration of the leech CNS.

As neurons grow to their targets their processes elongate, branch and form specialized endings into which are inserted appropriate ion channels. Our aim has been to analyse the role of the extracellular matrix molecules laminin and tenascin in inducing growth and in determining the form and physiological properties of growing neurites. A preparation in which development and regeneration can be followed at the cellular and molecular level in the animal and in tissue culture is the central nervous system (CNS) of the leech. In leech extracellular matrix (ECM) both laminin and tenascin are present; the molecules are structurally similar but not identical to their vertebrate counterparts. Tenascin extracted from leech ECM shows a typical hexabrachial structure whereas laminin shows a typical cruciform structure in rotary shadowed preparations. Leech laminin purified by means of a monoclonal antibody is a molecule of about 1000 kDa, with a polypeptide composition of 340, 200, 180 and 160 kDa. Substrates that contain tenascin or laminin produce rapid and reliable outgrowth of neurites by identified cells. A remarkable finding is that the outgrowth pattern produced by an individual neuron depends in part on its identity, in part on the substrate upon which it is placed. For example, a Retzius cell grows in a quite different configuration and far more rapidly on laminin substrate than does another type of neuron containing the same transmitter (serotonin); and the pattern of outgrowth of the Retzius cell is different on laminin and on the plant lectin Con A (concanavalin A). Thus Con A induces the growth of processes that are shorter, thicker, more curved and contain fewer calcium channels than those grown on laminin. To determine whether laminin can also influence neurite outgrowth in the animal, immunocytological techniques have been used to follow its distribution in the extracellular matrix of normal, developing and regenerating leech CNS. In adult leeches neuronal processes in the CNS are not in contact with laminin which is confined to the surrounding extracellular matrix. In embryos however, laminin staining appears between ganglionic primordia along the pathways that neurons will follow. Similarly, after injury to the adult CNS, laminin accumulates at the very sites at which sprouting and regeneration begin. How the laminin becomes redistributed to appear in the region of injury has not yet been established. Together these findings suggest a key role for laminin and for other extracellular matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of laminin and microglial cells at sites of injury and regeneration in the central nervous system of the leech.

Profuse sprouting of leech neurons occurs in culture when they are plated on a substrate consisting of laminin molecules extracted from extracellular matrix that surrounds the central nervous system (CNS). To assess the role of laminin as a potential growth-promoting molecule in the animal, its distribution was compared in intact and regenerating CNS by light and electronmicroscopy, after it had been labelled with an anti-leech-laminin monoclonal antibody (206) and conjugated second antibodies. In frozen sections and electron micrographs of normal leeches the label was restricted to the connective-tissue capsule surrounding the connectives that link ganglia. Immediately after the connectives had been crushed the normal structure was disrupted but laminin remained in place. Two days after the crush, axons began to sprout vigorously and microglial cells accumulated in the lesion. At the same time, labelled laminin molecules were no longer restricted to the basement membrane but appeared within the connectives in the regions of neurite outgrowth. The distribution of laminin at these new sites within the CNS was punctate at two days, but changed over the following two weeks: the laminin became aggregated as condensed streaks running longitudinally within the connectives beyond the lesion. The close association of regenerating axons with laminin suggests that it may promote axonal growth in the CNS of the animal as in culture.