Dkk1 and Dkk2 regulate epicardial specification during mouse heart development

Abstract

Background: Dkk1 and Dkk2 interact with LRP5 and LRP6 to modulate canonical Wnt signaling during development, and are known to be expressed in the developing heart. However, a loss-of-function mutation in either gene by itself produces no discernable heart phenotype.

Methods: Using standard husbandry techniques, Dkk1 null and Dkk2 null mouse lines were crossed to create double null embryos, which we examined using histological and immunohistochemical methods.

Results: Double null embryos die perinatally, with a gross head phenotype reminiscent of Dkk1 null embryos. Upon examination of late stage hearts, we observe myocardial defects including ventricular septal defects. At earlier stages, double mutant hearts show myocardial and epicardial hyperplasia. Myocardial hypertrophy is associated with a moderate increase in cell proliferation, but epicardial hypercellularity is not. Rather, the field of proepicardial precursor cells near the liver shows a broadening of expression for the cardiac-specific gap junction protein Connexin 43.

Conclusions: Dkk1 and Dkk2 both inhibit Wnt signaling to regulate early myocardial proliferation and each can compensate for the loss of the other in that role. Wnt signaling regulates myocardial proliferation in both heart fields at early stages. Additionally, Wnt signaling is sufficient to increase proepicardial specification as measured by Connexin 43 expression, resulting in a hypercellular epicardium and perhaps contributing to later defects.

Figure 1

Dkk1, Dkk2 double null embryos have cardiac defects at late developmental stages. A, B - Control and mutant heart sections at E18.5 reveal multiple defects including hypertrophic myocardium, trabeculae, and ventral septal defect. C, D - Magnified areas of interest shown by boxes in (A, B) respectively. In these close-ups the myocardial thickening is more pronounced, while endocardium and epicardium appear normal at this stage. E, F - Control and mutant heart sections at E15.5 reveal similar defects including hypertrophic myocardium and trabeculae. These sections are from a similar depth in the heart. G, H - Magnified areas of interest shown by boxes in (E, F) respectively. In these close-ups the myocardial thickening is pronounced. The endocardium and epicardium appear normal at this stage. Mutant refers to the double null genotype, while Control refers to either genetically normal (Dkk1 and Dkk2^+/+) animals or heterozygous siblings which are indistinguishable from ^+/+ animals.

Figure 2

Dkk1, Dkk2 double null embryos have cardiac defects at middle and early developmental stages. A-C - Heart of a control E12.5 embryo at the level of the outflow tract (A) or atrioventricular endocardial cushion (B), and magnified area of myocardium (C) indicated in the boxed area shown in (B). D-F - Heart of a Dkk1, Dkk2 double null E12.5 embryo at the level of the outflow tract (D) or atrioventricular endocardial cushion (E), and magnified area of myocardium (F) indicated in the boxed area shown in (E). The ventricular myocardium appears only modestly thicker in the mutant at this stage. However, the trabeculae are noticeably full and hyperplastic compared to the control. The epicardial cells in the mutant appear more closely spaced than to the control, and are occasionally multilayered (arrowheads). Both endocardial cushions also appears hypercellular and larger than the controls. G-I - Heart of a control E10.5 embryo at the level of the outflow tract (G) or atrioventricular endocardial cushion (H), and magnified area of myocardium (I) indicated in the boxed area shown in (H). J-L - Heart of a Dkk1, Dkk2 double null E10.5 embryo at the level of the outflow tract (J) or atrioventricular endocardial cushion (K), and magnified area of myocardium (L) indicated in the boxed area shown in (K). The ventricular and trabecular myocardium appear thick and hypercellular in the mutant compared to the control heart. The mutant epicardium is clearly multilayered (compare I, L, arrowheads). Both cushions also appear to be hypercellular and have larger areas than the controls.

Figure 3

Dkk1 and Dkk2 transcripts are expressed in an asymmetrical pattern through stage E15.5 in normal hearts. A-B - An E12.5 heart stained for Dkk1 transcripts, ventral (A) or dorsal (B) aspect. In the ventral aspect the right ventricle is on the left. The pattern of staining appears much stronger in the left ventricle but is visible in the right ventricle. Atrial staining is very faint. C-D - An E12.5 heart stained for Dkk2, ventral (C) or dorsal (D) aspect. The staining pattern appears almost identical to that of Dkk1. E-F - An E15.5 heart stained for Dkk1 transcripts, ventral (E) or dorsal (F) aspect. The staining pattern of Dkk1 is quite similar to that in E12.5 embryos, but the atrial staining appears more pronounced, especially in the right atrium. G-H - An E15.5 heart stained for Dkk2 transcripts, ventral (G) or dorsal (H) aspect. The staining pattern of Dkk2 is quite similar to that in E12.5 embryos, but the atrial staining appears more pronounced.

Figure 4

Dkk1, Dkk2 double null embryonic hearts express correct differentiation markers but contain ectopic multipotent cells. A-B - Control (A) and Dkk1, Dkk2 double mutant (B) hearts at E12.5 stained for the myocardial marker MF-20 (red). C-D - Control (C) and Dkk1, Dkk2 double mutant (D) hearts at E10.5 stained for the epithelial marker pan-cytokeratin (brown). Note the thickness and multilayering of the epicardium near the bottom of the mutant image (arrowheads). In control and mutant sections a few cells on the edge of the myocardium are also positive for cytokeratin suggesting epicardial origin. E-F - Control (E) and Dkk1, Dkk2 double mutant (F) hearts at E10.5 stained for the multipotency marker c-Kit (brown). Note the large cluster of c-Kit-positive cells in the mutant epicardium near the top of (F, arrowhead). In this system the blood cells also stain brown (marked with asterisks) but do not indicate the presence of multipotent stem cells. Blood cells can be identified through position and morphology.

Figure 5

Dkk1, Dkk2 double mutant embryos show increased myocardial proliferation and epicardial specification. A-B - Control (A) and mutant (B) embryonic hearts stained for phospho-Histone H3 reveal more positive cells in the mutant myocardium, but not epicardium. The epicardium is marked by white arrowheads, while the myocardium consists of the cells inside the epicardial layer. These results were consistent in multiple subjects. Note that blood cells in this system appear to be stained, but faintly. Blood cells can be identified by position and morphology. C-D - Control (C) and mutant (D) embryonic hearts stained with the in situ cell death system (TUNEL, green) show very few cells in either subject to be stained. These results were consistent in multiple subjects. E-F - Control (E) and mutant (F) embryonic hearts stained for the multipotent marker CX43 (green) show a dramatically increased field of expression in the mutant, apparently extending into the developing liver (region marked by asterisks).