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Monoclonal antibodies were raised against differentiated cells, and blastemal cells from regenerating limbs of adult newts (Notophthalmus viridescens) and screened for specific staining by immunocytochemistry. In addition to antibodies that identify muscle, Schwann cells and cartilage, two reagents were specific for subpopulations of blastemal cells. One of these latter antibodies, termed 22/18, has provided new evidence about the origin of blastemal cells from Schwann cells and myofibres, and also identifies blastemal cells whose division is persistently dependent on the nerve supply.
Fig. 1. Staining of the normal limb with myofibre and myelin markers. Methacrylate
sections were stained with a myofibre marker, 12/101, and a myelin marker, 30/26, as
described in Materials and Methods. (A) A region of the limb containing muscle (left
field) and the nerve sheath (right field) viewed under phase-contrast optics (bar =
30 jim) and (B) rhodamine optics to show staining of myofibres with 12/101.
(C) Approximately the same region as in A and B on an adjacent section viewed under
phase-contrast optics (bar = 35 jum) and (D) rhodamine optics to show staining of the
myelin sheaths with 30/26.
Fig. 2. Schematic diagram of 22/18+ and 22/31+ cells in the mature and regenerating
forelimb. The occurrence of 22/18+ cells (closed circles) and 22/31+ cells (open circles)
in dermis, nerve, bone, muscle and the blastema in (A) an unamputated, mature limb,
(B) a regenerating limb at wound healing, (C) a regenerating limb at the early- to
medium-bud stage, or (D) a regenerating limb at the late-bud stage.
Fig. 3. 22/31 and 22/18 staining at early-bud stages. Tissue sections of an early-bud,
forelimb blastema were stained with both 22/18 and 22/31 as described in Materials
and Methods using techniques to separately detect 22/18 binding with rhodamine and
22/31 binding with fluorescein. (A) A field in the middle of the blastema photographed
under Nomarski optics, and (B) rhodamine optics to detect 22/18 staining or (C)
fluorescein optics to detect 22/31 staining. Note that only a few cells stain with 22/31
while a majority stain with 22/l8. Furthermore, the 22/31"1" cells do not stain, or stain
very lightly, with 22/18. (D) A field in the tip of the blastema underneath the wound
epidermis photographed with Nomarski optics, or (E) rhodamine optics to detect
22/18 staining or (F) fluorescein optics to detect 22/31 staining. The left side of this
field contains epidermis that does not stain with either antibody. Of the blastemal cells
in this field, some stain with just 22/18, some with just 22/31 and some with both.
Bar = 5 0 ^ .
Fig. 4. 22/18 and 22/31 staining at late-bud stage. Longitudinal sections of a late-bud,
forelimb blastema were stained with both 22/18 and 22/31 as described in the legend to
Fig. 3. (A) A field at the tip of the blastema underneath the wound epidermis
photographed with Nomarski optics, or (B) rhodamine optics to detect 22/18 staining
or (C) fluorescein optics to detect 22/31 staining. Note that many of the blastemal cells
stain with 22/31 while only a subpopulation stains with 22/18. (D),(E) and (F) A field
in a tissue section of a different late-bud-stage blastema photographed in the same way
as in A,B and C. Note that 22/18 only stains a subpopulation of blastemal cells and
these cells (arrow in E) are mainly 22/31+. (Bars = 50//m.)
Fig. 5. 22/18 and myelin staining in the early-bud stage. Longitudinal tissue sections of
an early-bud, forelimb blastema were stained with both 22/18 and with 30/3,30/14 and
30/26. (A) A region of the limb between the blastema and mature tissue was
photographed under Nomarski optics (bar = 20jum), (B) fluorescein optics to detect
the Po antibodies or (C) rhodamine optics to detect 22/18 staining. In this photograph,
the nerve runs from mature tissue on the left to the blastema on the right. Note the
presence of cells that stain with both 22/18 and the Po antibodies.
Fig. 6. 22/18 and myofibre staining in the palette-stage regenerate. Transverse tissue
sections of a palette-stage regenerate were stained with both 22/18 and 12/101 as
described in Materials and Methods. (A) A section of the regenerate containing a
region of muscle differentiation was photographed under Nomarski optics (bar =
50 jum) with epidermis in the upper field and newly formed cartilage in the lower field.
(B) Same region photographed with rhodamine optics to detect 22/18 staining and (C)
fluorescein optics to detect 12/101 staining. Note that an area where 22/18 staining
occurs is a site of 12/101 staining (large arrow). (D,E and F) are photographed under
the same conditions but at a higher magnification (bar = 20jum).
