XB-ART-27369Dev Biol August 1, 1988; 128 (2): 441-52.
Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis.
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 tadpole tail 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 tail muscle cells and taken up by phagocytic cells.
PubMed ID: 3294065
Article link: Dev Biol
Genes referenced: acta1 acta4 actb ocm3 ocm4.2 ocm4.5
Antibodies: Acta1 Ab1 FITC-phalloidin Lectin Ocm Ab1
Article Images: [+] show captions
|FIG. 1. Western blot of various tissues probed with the PV antibody. Lanes (A-E) contain equivalent amounts of protein extracted from stage 55 tadpole tail, adult brain, adult mylohyoid muscle, adult gastrocnemius muscle, and adult sartorius muscle, respectively. The adult tissues were from a malexenqpus. The molecular weights of size standards are labeled on the left in kilodaltons &Da).|
|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 tadpole tail (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 tadpole tail, 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 tadpole tail. (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 tail muscle. (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 tadpole head, 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).|