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Abstract
We have used immunocytological techniques to examine the developmental expression of the Ca2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpoletail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tailmuscle cells and taken up by phagocytic cells.
FIG. 2. Immunolocalization of PV in different staged Xe-nop~~ embryos. Stage (A) 23, (B) 24, (C) 25, (D) 2’7, (E) 31, and (F) 55 embryos
(Nieuwkoop and Faber, 1967) were preserved in Bouin’s fixative, embedded in paraffin, and cross-sectioned in the midportion of the embryo
(A-E) or the tadpoletail (F). Rabbit anti-parvalbumin antibodies were detected with goat anti-rabbit IgG antibodies conjugated to FITC. Due
to the abundance of yolk in frog embryos, there is a high level of autofluorescence in the sections of stage 23-24 embryos. In (E) some structures
are labeled (C, neural tube (CNS); N, notochord; S, somite, G, gill arches; E, endoderm). Bar = 100 pm in (A-E) and 250 pm in (F).
FIG. 3. Organization of muscle fiber types in tadpoletail, Tails from stage 41 embryos were fixed, permeablised, and stained with either
phalloidin or the PV antibody. (A) The tail in longitudinal section in phase-contrast, the hindportion of the brain is labeled (Br). (B) The same
section stained for PV immunofluorescence. (C) PV-stained whole mount of a strip of fibers teased from the caudal portion of stage 41 tadpoletail. (D) Phase-contrast micrograph of a comparable tail strip; the black dots are melanocytes. (E) Rhodamine-conjugated phalloidin staining
of a tail strip. (F) PV immunoflourescence staining of s
FIG. 4. Fiber type composition of tailmuscle. (A) Phase-contrast image of a crosssection through a stage 55 embryo at the anterior end of the
tail. (B) The identical section stained with a mouse monoclonal antibody (JLA20) which reacts with skeletal actin. (C) PV immunoreactive
fibers within the same section. The arrows in (A-C) highlight a single muscle cell at the outer rind of the tail which reacts with phalloidin, but
does not react with PV antibodies. (D) bright-field view of a cryostat sectioned stage 55 tail that has been stained for NADH tetrazolium
reductase, an enzyme activity which is typically restricted to slow-twitch muscle fibers. In (D), the histochemical deposits are restricted to the
muscle cells at the outer rind of the tail. Bar = 38 pm.
FIG. 5. Immunofluorescence staining for PV in degenerating stage 64 tadpole tails. (A) Phase-contrast image of the myotomal muscles from
the middle region of the tail. (B) The same section stained for PV. (C) Phase-contrast image of a strip of muscle from the caudal end of the tail,
with two dying PV+ cells, of which the upper one is advanced in its degeneration. (D) Corresponding PV immunofluorescence. The black
star-like objects in (A) and (C) are melanocytes, and the black column in (C) is a blood vessel. (E) Phase-contrast view of a cross-section of a
caudal portion of the tail. (F) Corresponding PV immunofluorescence staining. Bar = 38 pm in (A) and (B), 19 pm in (C) and (D), and 75 pm in
(E) and (F).
FIG. 6. Immunofluorescence staining in the tadpole mylohyoid muscle. (A) Whole mount preparation of a fixed stage 41 mylohyoid muscle.
(B) The same muscle stained with parvalbumin antibodies; there are more immunoreactive fibers in the posterior half than in the anterior half
of the mylohyoid. (C) phase-contrast section through a mylohyoid muscle in a stage 41 tadpolehead, with the corresponding immunofluorescence
staining for parvalbumin in (D) which has been overexposed in the posterior half of the mylohyoid. Bar = 152 pm in (A) and (B) and 38 nm
in (C) and (D).
FIG. 7. Immunofluorescence staining of hindlimb from a stage 55 embryo. (A) phase-contrast image through the knee. (B) PV+ cells from the
same section. (C) phase-contrast micrograph of a region of a hindlimb with its cartilage. (D) PV staining of the same section. Bar = 190 pm in
(A) and (B) 38 pm in (C) and (D).
