Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Abstract

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues.

Fig. 1

Western blot detection of Hsp27 and Hsp70 in zebrafish embryos. a Developmental time course of Hsp27 expression. Numbers are time of sample harvest in hours postfertilization (hpf). b Concentration effects of scrambled (scr) and antisense hsp27 (hsp27i) PMO injection on Hsp27 expression. Numbers represent injection concentration in millimolar. A sample from uninjected embryos (uninj) is also shown. c Effects of 0.15-mM hsp27i PMO injection on Hsp27 expression in embryos of varying ages. The expression of sarcomeric (sarc) actin is shown for comparison. d Effects of 0.1 mM hsp27i PMO injection on expression of constitutive Hsc70 and inducible Hsp70 in control (CN) and heat-shocked (HS) embryos. Samples were harvested at 36 hpf. All lanes were loaded with equal amounts (30 μg) of total embryo protein

Fig. 2

Immunolocalization of Hsp27 in 50-hpf zebrafish embryos. All images were obtained with identical illumination and camera settings. Top row: Hsp27 expression is detected in the head (left), trunk (middle), and heart (right) of 0.1 mM scrambled (scr)-PMO-injected embryos under control conditions (CN). Hsp27 is most highly expressed in craniofacial muscles (arrows, left, and inset), in myocytes near the developing lateral line of the trunk (center, arrow), and in the heart (right). Middle row: Hsp27 expression increases in all tissues of 0.1 mM scr-PMO-injected embryos after heat shock (HS). Bottom row: Hsp27 immunostaining of embryos injected with 0.1 mM antisense hsp27 (hsp27i) PMO and heat-shocked. The inset images in the bottom row (not to scale) were digitally enhanced to show the section morphology. Images show the dorsal surface up for head and trunk images and down for heart images. Scale bars are 50 μm

Fig. 3

Morphology of zebrafish embryos is not altered by Hsp27 knockdown. a Overall morphology of 24-hpf embryos after injection at the one to two cell stages with 0.15-mM scrambled (scr) or antisense hsp27 (hsp27i) PMO. Scale bar = 1 mm. b Tail length of embryos measured from the tip of the tail to the yolk mass (arrows, a). c Lens diameter. d Two-dimensional area of the yolk. Values were obtained from 20 or more embryos in each group collected in a total of three independent trials

Fig. 4

Striated muscle architecture and heart rate in 50-hpf embryos are not altered by Hsp27 knockdown. a Phalloidin staining of skeletal muscle in embryos. These images are centered on the 12th myotome. b Area of the 12th myotome measured from 30 embryos injected with 0.1 mM scrambled (scr) or antisense hsp27 (hsp27i) PMO in a total of three independent trials. c Z-series projections of F-actin in hearts of embryos injected with hsp27i or scr PMO. Images are representative of four projections in each group. d Heart beat frequency measured from 40 scr and 40 hsp27i-PMO-injected embryos. No significant differences in myotome area or heart rate (p ≥ 0.05) were detected. Error bars are standard deviations. Scale bars are 50 μm (myotomes) and 10 μm (hearts)

Fig. 5

Motility of 50-hpf embryos is not altered by Hsp27 knockdown. a Traces of motility by embryos injected with 0.1 mM scrambled (scr) or antisense hsp27 (hsp27i) PMO. Traces are representative of at least 30 embryos per group measured in three independent trials. Axes show x (horizontal) and y (vertical) image coordinates. b Mean maximal velocity of hsp27i and scr-PMO-injected embryos in millimeter per second. No statistically significant (p ≥ 0.05) difference was detected. Error bars indicate standard deviations

Fig. 6

Hsp27 immunolocalization in unstressed adult zebrafish tissues. Hsp27 was detected throughout the heart myocardium (Hrt) and skeletal muscle, especially in slow-twitch muscle and at the boundary between external slow and internal fast-twitch muscle (arrow, Ske). Hsp27 was detected within the smooth muscle surrounding the intestinal tract (arrow, Int) and in the lining of the brain tissues (arrow, Br). Hsp27 was also detected at the margin of seminiferous tubules in the testis (Test). Right-hand panels are secondary antibody-only controls of each tissue type. Scale bars are 50 μm. A Western blot probed for Hsp70 and Hsp27 expression in white, or fast-twitch, muscle (wm), red, or slow-twitch, muscle (rm), brain, heart, lens, testis, and gut tissues is shown for comparison