Observations of the living developing nerve fiber

TL;DR

Harrison isolated fragments of frog embryonic nervous tissue in clotted adult frog lymph and observed living nerve fibers extending into the clot, showing that the fiber grows by the outflowing of protoplasm whose distal end retains amoeboid activity. This provided direct evidence that the axis cylinder is drawn out by amoeboid movement of the nerve cell's protoplasm, without participation of other cells.

Key findings

• Fibers leaving the nerve-tissue mass into the surrounding lymph-clot were usually first seen 2, occasionally 3, and once or twice 4 days after isolation; they are almost hyaline protoplasm devoid of yolk granules, about 1.5–3 µ thick, with irregular varicosities along their contours.
• The most remarkable feature is the enlarged end, from which extend numerous fine simple or branched filaments resembling rhizopods, with continual and rapid changes in form — "a mass of protoplasm undergoing amoeboid movements."
• When observed before growth was complete, the active end drew out and lengthened the fiber: one fiber lengthened almost 20 µ in 25 minutes, another over 25 µ in 50 minutes; the longest fibers observed were 0.2 mm.
• Branchial placodal thickenings formed similar fibers (in several cases seen to arise from individual cells), whereas myotomes, yolk endoderm, notochord, and indifferent ectoderm of the abdominal region did not — indicating a specific characteristic of nervous tissue.
• In embryos in which a segment of medullary cord was replaced by a cylindrical blood/lymph clot, serial sections after 2–4 days showed funicular fibers from the brain and anterior cord (naked axones without sheath cells) had grown a considerable distance into the clot, confirming that the fiber develops by outflowing of protoplasm from central cells, with no other cells or living structures taking part.

Methods

• Pieces of embryonic tissue known to give rise to nerve fibers (whole or fragments of the medullary tube, or ectoderm from the branchial region) were dissected from frog embryos about 3 mm long, shortly after closure of the medullary folds, when no visible differentiation of nerve elements is present.
• Tissue was transferred by fine pipette to a cover slip bearing a drop of lymph freshly drawn from an adult frog lymph-sac; the lymph clots quickly, fixing the tissue, and the cover slip was inverted over a hollow slide with the rim sealed with paraffine. With reasonable aseptic precautions, tissues lived a week and in some cases nearly four weeks, and could be observed daily under high magnification.
• For permanent preparations, a piece of medullary cord about four or five segments long was excised and replaced by a cylindrical clot of adult frog blood or lymph clotted in a capillary tube; after 2, 3, or 4 days specimens were preserved and examined in serial sections.

Limitations

• Stated: the cell aggregates making up organs do not undergo normal transformation in form, owing in part to abnormal conditions of mechanical tension, though individual tissue elements differentiate characteristically.
• Stated: because fibers develop largely within the mass of transplanted tissue itself, their mode of development cannot always be followed.
• Stated: permanent specimens showing isolated living fibers completely intact could not be made — the structures are so delicate that mere immersion in preserving fluid causes violent tearing, often tearing the tissue entirely from the clot; sections of some specimens showed nerves traced from the medullary tube walls but broken off short.