Ellen A.G. Chernoff

Regeneration of the urodele limb: A review

Urodele amphibians have been widely used for studies of limb regeneration. In this article, we review studies on blastema cell proliferation and propose a model of blastemal self‐organization and patterning. The model is based on local cell interactions that intercalate positional identities within circumferential and proximodistal boundaries that outline the regenerate. The positional identities created by the intercalation process appear to be reflected in the molecular composition of the cell surface. Transcription factors and signaling molecules involved in patterning are discussed within the context of the boundary/intercalation model. Developmental Dynamics 226:280–294, 2003.

Urodele spinal cord regeneration and related processes

Urodele amphibians, newts and salamanders, can regenerate lesioned spinal cord at any stage of the life cycle and are the only tetrapod vertebrates that regenerate spinal cord completely as adults. The ependymal cells play a key role in this process in both gap replacement and caudal regeneration. The ependymal response helps to produce a different response to neural injury compared with mammalian neural injury. The regenerating urodele cord produces new neurons as well as supporting axonal regrowth. It is not yet clear to what extent urodele spinal cord regeneration recapitulates embryonic anteroposterior and dorsoventral patterning gene expression to achieve functional reconstruction. The source of axial patterning signals in regeneration would be substantially different from those in developing tissue, perhaps with signals propagated from the stump tissue. Examination of the effects of fibroblast growth factor and epidermal growth factor on ependymal cells in vivo and in vitro suggest a connection with neural stem cell behavior as described in developing and mature mammalian central nervous system. This review coordinates the urodele regeneration literature with axial patterning, stem cell, and neural injury literature from other systems to describe our current understanding and assess the gaps in our knowledge about urodele spinal cord regeneration. Developmental Dynamics 226:295–307, 2003.

Extending the Table of Stages of Normal Development of the Axolotl: Limb Development

The existing table of stages of the normal development of the axolotl (Ambystoma mexicanum) ends just after hatching. At this time, the forelimbs are small buds. In this study, we extend the staging series through completion of development of the forelimbs and hindlimbs. Developmental Dynamics 226:000–000, 2003.

Matrix metalloproteinase production in regenerating axolotl spinal cord.

In urodele amphibian spinal cord regeneration, the ependymal cells lining the central canal remodel the lesion site to favor axonal regrowth. We profiled the production of matrix metalloproteinases by injury‐reactive mesenchymal ependymal cells in vivo and in vitro and found that matrix metalloproteinases are involved in this remodeling process in the axolotl (Ambystoma mexicanum). The production of cell‐associated matrix metalloproteinases in vivo was shown to be identical to that in our cultured ependymal cell model system. Activated and zymogen forms of matrix metalloproteinases were identified using zymography, chemical inhibitors of matrix metalloproteinases, and cleavage of propeptides by organomercurials. The principal cellular proteinases consisted of matrix metalloproteinase‐2 (gelatinase A) and matrix metalloproteinase‐1 (type I collagenase), which display characteristic shifts in molecular weight following proenzyme processing by organomercurials. In addition, ependymal cell conditioned medium contained secreted forms of the enzyme undetectable in situ. Matrix metalloproteinase‐9 (gelatinase B) as well as matrix metalloproteinase‐2 and matrix metalloproteinase‐1 were secreted and casein substrate zymography showed the presence of a small amount of a very high molecular weight matrix metalloproteinase‐3 (prostromelysin) secreted into the culture medium. Matrix metalloproteinases were still present at 4 weeks post‐lesioning when the ependymal cells have just re‐epithelialized, but decreased near the completion of regeneration (8 weeks post‐lesioning). Zymography showed no detectable matrix metalloproteinases in unlesioned cord but the presence of tissue inhibitor of metalloproteinase‐1 in intact cord was seen by Western blotting. This study shows that matrix metalloproteinases are associated with urodele spinal cord regeneration and validates the use of our ependymal cell tissue culture model system to evaluate ependymal cell behavior during spinal cord regeneration.

Matrix metalloproteinase expression during blastema formation in regeneration-competent versus regeneration-deficient amphibian limbs.

We used an antibody array to compare the protein expression of matrix metalloproteinases (MMPs)‐1, ‐2, ‐3, ‐8, ‐9, ‐10, and ‐13, as well as the tissue inhibitors of metalloproteinases (TIMPs)‐1, ‐2, and ‐4 during blastema formation in amputated hindlimbs of regeneration‐competent wild‐type axolotls and stage‐54 Xenopus, and regeneration‐deficient short‐toes axolotls and Xenopus froglets. Expression of MMP‐9 and ‐2 was also compared by zymography. Both short‐toes and froglet failed to up‐regulate MMPs in a pattern comparable to the wild‐type axolotl, suggesting that subnormal histolysis is at least in part responsible for the poor blastema formation characteristic of both short‐toes and froglet. MMP levels were much lower in amputated stage‐54 Xenopus limb buds than in the other animals, suggesting that blastema formation in these limb buds requires much less extracellular matrix degradation than in fully differentiated limbs. TIMP expression patterns followed the same trends as the MMPs in each group of animals. Developmental Dynamics 240:1127–1141, 2011.

Developmental aspects of spinal cord and limb regeneration

The ability of birds and mammals to regenerate tissues is limited. By contrast, urodele amphibians can regenerate a variety of injured tissues such as intestine, cardiac muscle, lens and neural retina, as well as entire structures such as limbs, tail and lower jaw. This regenerative capacity is associated with the ability to form masses of mesenchyme cells (blastemas) that differentiate into the missing tissues or parts. Understanding the mechanisms that underlie blastema formation in urodeles will provide valuable tools with which to achieve the goal of stimulating regeneration in mammalian tissues that do not naturally regenerate. Here we discuss an example of tissue regeneration (spinal cord) and an example of epimorphic appendage regeneration (limb) in the axolotl Ambystoma mexicanum, emphasizing analysis of the processes that produce the regeneration blastema and of the tissue interactions and blastemal products that contribute to the regeneration‐promoting environment.

Effect of carbon nanoparticles on renal epithelial cell structure, barrier function, and protein expression

Abstract To assess effects of carbon nanoparticle (CNP) exposure on renal epithelial cells, fullerenes (C60), single-walled carbon nanotubes (SWNT), and multi-walled carbon nanotubes (MWNT) were incubated with a confluent renal epithelial line for 48 h. At low concentrations, CNP-treated cells exhibited significant decreases in transepithelial electrical resistance (TEER) but no changes in hormone-stimulated ion transport or CNP-induced toxicity or stress responses as measured by lactate dehydrogenase or cytokine release. The changes in TEER, manifested as an inverse relationship with CNP concentration, were mirrored by an inverse correlation between dose and changes in protein expression. Lower, more physiologically relevant, concentrations of CNP have the most profound effects on barrier cell function and protein expression. These results indicate an impact of CNPs on renal epithelial cells at concentrations lower than have been previously studied and suggest caution with regard to increasing CNP levels entering the food chain due to increasing environmental pollution.

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.

Primary culture of axolotl spinal cord ependymal cells

In order to examine the role of ependymal cells in the spinal cord regeneration of urodele amphibians, procedures were established to identify and culture these cells. Cell isolation and culture conditions were determined for ependymal cells from larval and adult axolotls (Ambystoma mexicanum). Dissociated cells prepared from intact spinal cords were cultured on fibronectin- or laminin-coated dishes. Dissociated cells attached more rapidly to fibronectin, but attached and spread on both fibronectin and laminin. Essentially pure populations of ependymal cells were obtained by removing 2 week old ependymal outgrowth from lesion sites of adult spinal cords. These ependymal outgrowths attached and grew only on fibronectin-coated dishes. Growth and trophic factors were tested to formulate a medium that would support ependymal cell proliferation. The necessary peptide hormones were PDGF, EGF, and insulin. TGF-beta(1) affected the organization of cell outgrowth. Initially, longterm culture required the presence of high levels of axolotl serum. Addition of purified bovine hemaglobin in the culture medium reduced the serum requirement. Outgrowth from expiants was subcultured by transferring groups of cells. Intrinsic markers were used to identify ependymal cells in culture. The ependymal cells have characteristic ring-shaped nucleoli in both intact axolotl spinal cords and in culture. Indirect immunofluorescence examination of intermediate filaments showed that ependymal cells were glial fibrillary acidic protein (GFAP) negative and vimentin positive in culture. Identification of dividing cells was made using (3)H-thymidine incorporation and autoradiography, and by the presence of mitotic figures in the cultured cells.

An ependymal cell culture system for the study of spinal cord regeneration

Successful regeneration of lesioned adult spinal cord in urodele (caudate) amphibians requires the action of injury‐responsive ependymal cells (ependymoglia). The epithelial‐to‐mesenchymal transformation of ependymal cells following transection of the salamander spinal cord and the subsequent reformation of an epithelial tube have been described previously. A complete tissue culture model system has now been devised to study mesenchymal ependymal cells, epithelial ependymal cells, and ependymal/neuronal interactions in vitro. Here, we review critical aspects of substrate and growth factor environments required to produce mesenchymal ependymal cells in culture and present the first culture system for epithelial salamander ependymal cells and central nervous system neurons suitable for cell‐cell interaction studies. Critical to ependymal epithelialization in culture is the removal of epidermal growth factor and addition of thrombin. Epithelialization occurs on tissue culture plastic as well as on permeable culture substrates. This culture system can now be used to determine the initial trigger for the ependymal response. A preliminary examination of ependymal/neuronal interactions shows that coculture of mesenchymal ependymal cells and central nervous system neurons prolongs survival of the neurons.