RARß2 expression is induced by the down-regulation of microRNA 133a during caudal spinal cord regeneration in the adult newt.

BACKGROUND: Adult urodele amphibians represent unique model organisms to study spinal cord regeneration. Trauma to the spinal cord induces an ependymal response, activating multipotent neural stem cells that contribute to the redifferentiation of both glia and neurons in the regenerate. The molecular events underlying this ependymal response are not completely understood, but likely involve coordinated global changes in gene expression. MicroRNAs and retinoid signaling are postulated to orchestrate these patterns of gene expression in response to trauma. Our objectives were to determine the roles played by some miRNAs as potential regulators of retinoid signaling in this process. RESULTS: We found that the expression levels of miRNAs 133a, 203, and 124a are dysregulated during the first 21 days post amputation (dpa). Interestingly, these miRNAs are expressed primarily within the ependymoglia. We have shown in vitro that a miR-133a mimic targets the 3' UTR of the newt RARß2 transcript. Importantly, upregulation of this mimic in vivo led to a significant decline in RARß2 protein at 14 dpa and inhibited regeneration. CONCLUSIONS: These data are the first to link miRNAs and retinoid signaling during spinal cord regeneration and provide support for miR-133a as an upstream regulator of RARß2 expression in this process.

Retinoic acid stimulates the synthesis of a novel heat shock protein in the regenerating forelimb of the newt.

Morphogenetic effects of retinoic acid (RA) on the urodele amphibian limb regenerate pattern have been well documented, but little is known regarding the mechanism of this action of RA at the molecular level. Since exogenous RA, at concentrations sufficient to cause proximalization, represents a significant stress to newts and has been shown previously to elicit increased synthesis of heat shock proteins (HSPs) in mouse embryo limb buds, we investigated the effects of this putative morphogen on the synthesis of members of the 70-kilodalton (70-kDa) stress protein family in amputated forelimbs of the newt Notophthalmus viridescens. Injection (i.p.) of RA in dimethyl sulfoxide (DMSO), at a dose sufficient to cause significant proximal-distal reduplication of the pattern in 50% of animals treated, resulted in increased synthesis and accumulation of a 73-kDa protein with a pI of approximately 6.75. The synthesis of this same protein is increased in limb tissues as a result of a brief 35 degrees C heat shock. This protein is electrophoretically distinct from the newt HSP 70 family members, displays a different partial peptide map, and shows no immunological cross-reactivity with an anti-human HSP 70 monoclonal antibody. It may be a member of a separate family of 70- to 73-kDa HSPs. Interestingly, the synthesis of this protein is increased and it is more abundant in control, proximal moderate-early bud stage regenerates at 6 days after i.p. injection of DMSO than in similarly treated distal regenerates. This protein is, in addition, increased in distal regenerates to proximal levels by a prior injection of RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of proteins potentially involved in proximal-distal pattern formation in the regenerating forelimb of the newt.

We have used high resolution two-dimensional gel electrophoresis to identify and characterize proteins that may represent products of genes involved in establishing positional information along the proximal-distal axis of the regenerating forelimb of the newt Notophthalmus viridescens. At least 24 proteins have been found whose synthesis and (or) abundance is increased in proximal (midstylopodial) regenerates relative to midzeugopodial (distal) regenerates at either of two regeneration stages, the early dedifferentiation and moderate bud stages. Four of these same proteins show an axial asymmetry at both stages. Ten distal-specific proteins were also identified, although only one was common to both stages. More significantly, 6 of these 34 proteins (molecular masses of 73, 73, 51.5, 44.0, 19.5, and 16.5 kilodaltons and isoelectric points of 6.70, 6.74, 6.0, 6.05, 5.9, and 6.98, respectively) are regulated to proximal levels by treatment of distal regenerates with retinoic acid (RA) at both stages. An additional five are proximalized by RA at only one regeneration stage. Since the effect of RA is to proximalize positional information in blastema cells, these 11 proteins represent gene products that could be involved in a biochemical cascade leading to the establishment of positional information in the regenerating limb along this axis.

A low-molecular weight chick brain-derived growth factor is mitogenic for cultured astroglia from the chick embryo.

Immunohistochemically defined astrocytes from the 10-day chick embryo were stimulated to incorporate increased levels of [3H]thymidine when a low-molecular weight peptide growth factor, chick brain-derived growth factor (CBGF), was added to the cultures. Treatment of these GFAP-positive astrocytes with 10 ng/ml CBGF in medium supplemented with 1% fetal bovine serum resulted in a 3.5-4-fold increase in [3H]thymidine incorporation when compared to astrocytes cultured in defined medium supplemented with 1% serum alone. CBGF had no effect on the survival, proliferation or differentiation of a number of other cell types from the 10-day chick embryo brain, including neurons and meningeal fibroblasts. CBGF was also ineffective as a mitogen for chick embryo skeletal muscle myoblasts, primary mouse embryo fibroblasts and one murine teratocarcinoma-derived cell line (STO). We suggest that CBGF might act as a mitogenic signal for astroglia during central nervous system development and repair.

Partial purification of a low-molecular-weight growth factor from chicken brain.

The regenerating amphibian limb serves as a useful model for studying factors influencing cell proliferation and differentiation. In particular, peripheral nerves are thought to provide a stimulus for growth of the blastema, presumably via the elaboration of an as yet unidentified neurotrophic factor. In the present study, pressure ultrafiltration coupled with chromatofocusing have proven to be effective methods of partially purifying a neurotrophic factor from adult chicken brains. This chick brain growth factor (CBGF) appears to be a heat-stable, basic peptide of low molecular weight (less than 6,000). It is a potent mitogen in vitro, at nanomolar concentrations, for both blastema cells and Swiss mouse 3T3 fibroblasts. CBGF is apparently distinct from other peptide mitogens and/or neuromodulators that have been reported to stimulate blastema growth in vivo and in vitro. These include substance P, FGF from bovine brain and pituitary, EGF, transferrin (sciatin), and spinal cord growth factor (SCGF). The possible relationship of CBGF to other neural regulatory molecules is discussed.

Noradrenaline and cyclic AMP--independent growth stimulation in newt limb blastemata.

Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.