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Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus.
Abstract Wnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. We have identified a second Xenopus Wnt11-Related gene (Wnt11-R) that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube.
Fig. 2. In situ hybridization analysis of Wnt11-R expression during Xenopus development. (A) Dorsal view of st. 13 embryo showing earliest Wnt11-R expression in the dorsal region of the developing somites (arrow). (B) Dorsal view at st. 22 showing transcripts in the somite and neural tube. (C) Transverse section at st. 22 shows that Wnt11-R transcripts are limited to the dorsal margin of the somite (arrow) and the dorsal neural tube. (D) Dorsal view at st. 26 showing Wnt11-R expression in the somites, branchial arches, neural tube, and brain. (E) Lateral view of st. 26 embryo. (F) Ventral view of st. 26 embryo. (G) Lateral view at st. 28 shows Wnt11-R in the heart myocardium (arrow). (H) Lateral view at st. 28 with expression of MHCα marking the precardiac region. (I) Ventral view of st. 28 embryo showing Wnt11-R expression in precardiac tissue. (J) Ventral view of st. 28 embryo stained for MHCα expression. (K) Lateral view at st. 35 showing Wnt11-R expression in the branchial arches, somites, neural tube, fin, and the heart. (L) Transverse section through the a medial region of a st. 35 embryo showing Wnt11-R transcripts in the dorsal margin of the somite, dorsal neural tube, and mesenchymal cells within the fin. (M–O) Transverse sections progressing from anterior to posterior regions through the heart tube of a st. 35 embryo showing Wnt11-R expression in the myocardium of the heart. Sections show the future outflow tract and ventricle (M), the ventricle (N), and the atria (O). (P) MHCα expression in section equivalent to that in O, showing expression throughout myocardial layer. (Q) Wnt11-R is expressed throughout the myocardium of the st. 45 heart, although expression is lower in atrial tissues. Atria and ventricular (vent) tissues are indicated. (R) MHCα expression in the st. 45 heart showing strong expression throughout the myocardium.
Fig. 4. Wnt11-R antisense morpholino oligomer inhibits translation from Wnt11-R-GFP fusion transcripts. (A) Diagram of the Wnt11-R/GFP fusion (tester) mRNA, indicating binding locations for antisense MO1 and MO2. The ability of MOs to inhibit translation from mRNAs containing Wnt11-R sequences was assayed by coinjecting 500 pg of synthetic tester mRNA with 15 ng of Wnt11-R MO1 into fertilized eggs. (B–D) At about stage 13, embryos were viewed under UV light or standard illumination. Uninjected embryos (B) serve as negative control and do not glow. Embryos injected with tester mRNA (C) fluoresce, confirming the translation of GFP protein. Embryos coinjected with MO1 plus tester mRNA (D) show strongly reduced fluorescence, indicating effective inhibition of translation. The second Wnt11-R antisense MO, MO2, showed no inhibition of GFP expression using this assay (data not shown). (E) Immunoblot detection of GFP protein in whole embryo extracts from uninjected embryos, tester mRNA-injected embryos, and embryos coinjected with tester mRNA and 15 ng of MO1. GFP protein is detected in the tester sample, but is not detectable when translation is inhibited by MO1. Ponceau S staining indicates equal loading of sample. (F) Embryo injected with 15 ng of control MO (contMO) assayed at st. 28 by in situ hybridization for MHCα, showing normal cardiac differentiation. (G) Embryo injected with 15 ng of MO1 also shows normal appearance of differentiation markers when assayed for MHCα transcripts.
Fig. 5. Characterization of myocardial defects observed in Wnt11-R-depleted embryos. In all cases, myocardial tissue is detected by in situ hybridization using MHCα probe. (A–D) One-sided injection of either contMO (A and B) or Wnt11-R MO1 (C and D) does not interfere with myocardial differentiation when assayed at st. 28. (E–F) Control MO-injected embryos assayed at st. 34 showing normal expression of MHCα when viewed laterally (E), ventrally (F), or in section G. (H–K) MO1-injected embryos assayed at st. 34 showing myocardial marker expression in lateral (H) and ventral (I) views. Sectioning of embryos through the heart region (J and K) shows abnormal morphology of the heart tube. (L–N) Isolated hearts assayed at st. 45. Normal heart morphology is illustrated in L and abnormal, partially bilaterally duplicated hearts from Wnt11-R-depleted embryos are shown in M and N. Atria (a), ventricle (v), and outflow tracts (oft) are indicated. (O) Section through the heart tube of a st. 34 embryo injected with MO1 on one side, as indicated. (P) Same section as O showing detection of cell nuclei using propidium iodide staining under UV light. The myocardial layer is outlined. Nuclei were counted to assay cell number in Wnt11-R-depleted and -untreated myocardial tissue.
Fig. 6. Characterization of myocardial defects in Wnt11-R-depleted and JNK-inhibited embryos. (A) Whole mount immunostaining of unmanipulated st. 30 embryo showing activated JNK (p-JNK) in the heart region (arrow). (B) Transverse section through heart region of st. 30 embryo showing p-JNK-positive cells in the myocardial layer. (C) Transverse section through heart region of p-JNK-stained embryo. Arrowheads indicate regions of positive staining. (D) Nuclear staining with propidium iodide of same section shown in C illustrating the p-JNK localization is perinuclear. (E–H) Comparison of media control and JNK inhibitor (SP600125)-treated embryos at st. 33. (E and G) Whole mount in situ hybridization shows normal initiation of MHCα expression in treated and untreated embryos. (F and G) Transverse sections through the heart region of MHCα-stained embryos show that inhibition of JNK activation produces morphological defects in heart tube formation. (I–L) Transmission electron micrographs of transverse plastic sections through the myocardial wall from unmanipulated control and treated embryos at st. 33. In all panels, the endocardial side of the tube is to the right. Prominent dark spots are yolk granules. In all cases, spaces between adjacent cells have been colored in yellow. (I) Untreated control heart shows tightly packed columnar cells in myocardial wall with little space between adjacent cells. (J) Wnt11-R MO1-treated embryos show thickened wall of myocardium and increased extracellular space. (K) DMSO media-treated control hearts show very little space between adjacent cells in myocardium. (L) JNK inhibitor (SP600125)-treated embryos show increased thickness of myocardial layer and more extracellular space relative to DMSO-treated controls.
mapk8( mitogen-activated protein kinase 8 ) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.