Small heat shock protein Hsp27 is required for proper heart tube formation

Abstract

The small heat shock protein Hsp27 has been shown to be involved in a diverse array of cellular processes, including cellular stress response, protein chaperone activity, regulation of cellular glutathione levels, apoptotic signaling, and regulation of actin polymerization and stability. Furthermore, mutation within Hsp27 has been associated with the human congenital neuropathy Charcot-Marie Tooth (CMT) disease. Hsp27 is known to be expressed in developing embryonic tissues; however, little has been done to determine the endogenous requirement for Hsp27 in developing embryos. In this study, we show that depletion of XHSP27 protein results in a failure of cardiac progenitor fusion resulting in cardia bifida. Furthermore, we demonstrate a concomitant disorganization of actin filament organization and defects in myofibril assembly. Moreover, these defects are not associated with alterations in specification or differentiation. We have thus demonstrated a critical requirement for XHSP27 in developing cardiac and skeletal muscle tissues.

FIG. 1

XHsp27 is a conserved member of the Hsp27 subfamily of proteins. (a) Protein sequence alignments of X. laevis Hsp27 with various Hsp27 orthologues. Alignment was performed using the GeneDoc program. Blue underline indicates conserved crystallin domain. Red underline indicates putative actin interacting domains. (b) Percent identity and similarity between Hsp27 orthologues. (c) Synteny between X. tropicalis, human, mouse, rat, and chick Hsp27 loci as revealed by Metazome. Hsp27 is indicated in black. Upstream and downstream genes are colored as indicated.

FIG. 2

XHsp27 is expressed in the gastrula, and developing skeletal and cardiac muscle. Whole mount in situ hybridization of X. laevis embryos using an antisense probe specific for XHsp27 at the indicated stages. (a) Dorsal is to the top. (b–i) Anterior is to the left. (j–m) Transverse sections through somitic (j, k) and cardiac (l, m) regions. Dorsal is to the top. b, body muscle; br, brain; h, heart; j, jaw muscle, m, myotome; mc, myocardium.

FIG. 3

HSP27 morpholinos specifically block translation in vitro. (a) Diagram depicting XHsp27 mRNA structure and morpholino-targeted region. (b) Western blot demonstrating translation inhibition in vitro using rabbit reticulocyte lysate. Reactions were incubated with the indicated total amounts of HA-Hsp27 mRNA with/without HSP27MO. TBX20MO was included as a negative control. HSP27MO was incubated with HA-Tbx20 mRNA to show specificity of the HSP27MO. HA-HSP27 and HA-TBX20 were visualized using anti-HA antibody, and SHP2 antibody was used as loading control. (c) Western blot demonstrating translation inhibition in vivo by coinjection of HA-Hsp27 mRNA with HSP27MO. Embryos were injected with the indicated amount of HSP27MO, ControlMO, and/or HA-Hsp27 mRNA. Embryos were collected at Stage 20. Western blotting was performed on lysate using anti-HA antibody. SHP2 antibody was used as loading control.

FIG. 4

Depletion of XHSP27 results in unfused or partially fused hearts. Whole-mount antibody staining using anti-myosin heavy chain α (MHC). Embryos were injected at the one-cell stage with either HSPMO or ConMO, fixed, and stained for MHC at the indicated stages. All views are ventral with anterior upwards. Arrows indicate separation between the two cardiac fields or developing hearts. a, atrium; i, inflow tract; o, outflow tract; v, ventricle.

FIG. 5

Specification and differentiation of cardiac and skeletal muscle appear unaltered in HSP27 morphants. Whole-mount in situ hybridizations using antisense probes against (a) Nkx2.5, (b) Tbx20, (c) Gata4, (d) Gata6, (e) Mlc1v′, and (f) Titin novex 3 (Tn3). Embryos were injected with either HSP27MO or ControlMO and fixed at the indicated stages. (a–f) Ventral views with anterior upward. (e, f) Stage 37 embryos shown laterally with anterior to the left. All markers analyzed appear normal between control and HSP27 morphant embryos.

FIG. 6

Depletion of XHSP27 results in actin disorganization in developing skeletal and cardiac muscle. Transverse 12 μm sections through the heart (a–l) and somite (m–x). All sections are shown with dorsal upward. Heart sections were immunostained for F-actin using phalloidin (a, d, g, j) and MHC using anti-MHC antibody (b, e, h, k). Overlays are shown in (c, f, i, l). Somite sections were immunostained for F-actin using phalloidin (m, p, s, v) and stained with DAPI to visualize the nuclei (n, q, t, w). Overlays are shown in (o, r, u, x).

FIG. 7

Hsp-depletion does not alter levels of actin. Western blot demonstrating actin levels in control and Hsp27-depleted embryos (Hsp27MO) at the indicated stages. α-tubulin antibody staining was used as a loading control.

FIG. 8

XHSP27 morphant ultrastructure analysis reveals a lack in myofibril assembly. Transmission electron microscopy of ventral myocardium in Stage 38 control and XHSP27 morphant hearts. (a–c) Ventral myocardium of ConMO injected embryo. (d–f) Ventral myocardium of XHSP27MO injected embryo. Cardiac muscle fibrils are shown pseudocolored in yellow. Inset in (c) shows magnification of myofibril structure and inset in (f) shows magnification of apparent myosin aggregates. (g, h) Muscle fibers within the myotome in Stage 38 ConMO injected embryos. (i, j) Muscle fibers within the myotome in stage 38 XHsp27 morpholino injected embryos. l, longitudinal myofibers section; t, transverse myofibers section; z, z-line.