A comparative study of gland cells implicated in the nerve dependence of salamander limb regeneration

Abstract

Limb regeneration in salamanders proceeds by formation of the blastema, a mound of proliferating mesenchymal cells surrounded by a wound epithelium. Regeneration by the blastema depends on the presence of regenerating nerves and in earlier work it was shown that axons upregulate the expression of newt anterior gradient (nAG) protein first in Schwann cells of the nerve sheath and second in dermal glands underlying the wound epidermis. The expression of nAG protein after plasmid electroporation was shown to rescue a denervated newt blastema and allow regeneration to the digit stage. We have examined the dermal glands by scanning and transmission electron microscopy combined with immunogold labelling of the nAG protein. It is expressed in secretory granules of ductless glands, which apparently discharge by a holocrine mechanism. No external ducts were observed in the wound epithelium of the newt and axolotl. The larval skin of the axolotl has dermal glands but these are absent under the wound epithelium. The nerve sheath was stained post-amputation in innervated but not denervated blastemas with an antibody to axolotl anterior gradient protein. This antibody reacted with axolotl Leydig cells in the wound epithelium and normal epidermis. Staining was markedly decreased in the wound epithelium after denervation but not in the epidermis. Therefore, in both newt and axolotl the regenerating axons induce nAG protein in the nerve sheath and subsequently the protein is expressed by gland cells, under (newt) or within (axolotl) the wound epithelium, which discharge by a holocrine mechanism. These findings serve to unify the nerve dependence of limb regeneration.

Fig. 1

Ultrastructure of glands under the wound epithelium (WE) of the newt limb blastema. (A) A gland that has formed under the WE at 12 days post-amputation. (B) A higher magnification image of the gland in (A). A single cell (asterisk) is discharging its contents into the extracellular space, whereas another cell (arrowed) has discharged its contents. (C) A more mature gland under the proximal WE. The morphology resembles the adult gland type and is associated with the progressive differentiation of the regenerate. (D) A higher magnification of the gland in (C) showing secretory granules with moderate electron density (dotted line) that have accumulated at the leading edge of the cell. N, nucleus. Scale bars: 20 μm (A and C) and 10 μm (B and D).

Fig. 2

Immunogold labelling of the newt anterior gradient (nAG) protein in dermal glands of a newt limb blastema. (A) Section of a gland after reaction with rabbit antibodies to nAG protein, followed by gold-labelled anti-immunoglobulin reagent. Gold particles are visible in the secretory granules and cytoplasm. (B) A higher magnification of the boxed area from (A) showing labelling of the endoplasmic reticulum (ER). (C) A high-magnification image of a granule (GR) showing gold labelling. (D) A section after reaction with a non-specific antibody shows little or no reaction with the gland cells. An occasional gold particle is visible (arrowed). Scale bars: 0.2 μm (A and D) and 0.5 μm (B and C).

Fig. 4

Scanning electron micrograph showing the surface features of an axolotl limb blastema. (A) Low-magnification view of an axolotl limb blastema. (B) Boxed area from (A), showing the surface morphology of the wound epithelium (WE). Note the nature of the smooth WE lacking any glandular openings. (C) A magnified area showing the boundary between the limb stump and regeneration blastema. The skin of the limb stump has ciliated glandular pores (arrows), whereas the WE lacks any glands. Scale bars: 100 μm (A and B) and 10 μm (C).

Fig. 3

Scanning electron micrograph of wound epithelium (WE) in the newt blastema. (A) Low-power scanning electron microscopy of a newt limb blastema at 12 days post-amputation. The dotted line indicates the boundary of the sample and conductive silver paint. (B) A higher magnification view of the boxed area from (A) showing the surface of the WE of the limb blastema. Unlike adult skin, the WE is devoid of any glandular openings. (C) An area showing the boundary between the limb stump and regeneration blastema. The dotted line demarcates the old skin from the WE. Note the presence of glandular openings in the old skin (arrows), which is entirely absent in the WE. Scale bars: 100 μm (A and B) and 20 μm (C).

Fig. 6

Localization of axolotl anterior gradient (aAG) protein in the axolotl limb blastema by indirect immunofluorescence. (A) Expression of aAG protein in a section of a blastema at 11 days post-amputation. Note the reactivity of the nerve sheath as well as cells in the wound epithelium and the general lack of reactivity in mesenchymal cells. (B) A higher magification image of the boxed area from (A). Note the characteristic reactivity with a subset of cells. (C) Higher magnification image of the nerve sheath from (A). (D) A composite overlay of the boxed area in (A) using differential interference contrast and the nuclear stain Hoechst 33258. The arrows identify the cells expressing aAG protein in (B). M, mesenchyme; N, nerve. Scale bars: 200 μm (A) and 50 μm (D).

Fig. 5

Axolotl anterior gradient (aAG) protein. (A) Alignment showing the amino acid sequence of the aAG protein with newt anterior gradient protein. Highlighted amino acids represent the two peptides, 398 and 399, which were used as immunogens to raise antibodies in rabbits. (B) A section of nerve bundles in an axolotl forelimb (arrows) after reaction with the 398 antibody and detection by indirect immunofluorescence. Note the specific reactivity of the antibody to nerve bundles. (C) Immunoreactivity of a non-specific rabbit antibody reacted at a matched concentration to (B) in a cross-section of axolotl forelimb. N, nerve. Scale bars: 200 μm (B and C).

Fig. 7

Localization of axolotl anterior gradient (aAG) protein and serotonin in Leydig cells of the wound epithelium by double fluorophor immunofluorescence. (A) Expression of aAG protein detected with rabbit antibody 398. (B) Expression of serotonin in Leydig cells detected with rat monoclonal anti-serotonin. (C) Composite overlay showing that serotonin-positive Leydig cells express aAG protein. (D) Composite overlay of differential interference contrast and nuclear dye showing morphological features of the wound epithelium. WE, wound epithelium; M, mesenchyme. Scale bars: 200 μm (A–D).

Fig. 8

Effect of denervation on expression of axolotl anterior gradient (aAG) protein in the axolotl wound epithelium. (A) Section of the wound epithelium from an axolotl blastema at 9 days post-amputation showing aAG protein-positive cells (arrowheads) detected by indirect immunofluorescence. (B) Section of the blastema from the contralateral limb denervated 72 h prior to amputation. Note the absence of positive cells in the wound epithelium. WE, wound epithelium; M, mesenchyme. Scale bars: 200 μm (A and B).