Retinoic acid deficiency alters second heart field formation

Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development. The retinaldehyde dehydrogenase 2 (RALDH2) enzyme catalyzes the second oxidative step in RA biosynthesis and its loss of function creates a severe embryonic RA deficiency. Raldh2(-/-) knockout embryos fail to undergo heart looping and have impaired atrial and sinus venosus development. To understand the mechanism(s) producing these changes, we examined the contribution of the second heart field (SHF) to pharyngeal mesoderm, atria, and outflow tract in Raldh2(-/-) embryos. RA deficiency alters SHF gene expression in two ways. First, Raldh2(-/-) embryos exhibited a posterior expansion of anterior markers of the SHF, including Tbx1, Fgf8, and the Mlc1v-nlacZ-24/Fgf10 reporter transgene as well as of Islet1. This occurred at early somite stages, when cardiac defects became irreversible in an avian vitamin A-deficiency model, indicating that endogenous RA is required to restrict the SHF posteriorly. Explant studies showed that this expanded progenitor population cannot differentiate properly. Second, RA up-regulated cardiac Bmp expression levels at the looping stage. The contribution of the SHF to both inflow and outflow poles was perturbed under RA deficiency, creating a disorganization of the heart tube. We also investigated genetic cross-talk between Nkx2.5 and RA signaling by generating double mutant mice. Strikingly, Nkx2.5 deficiency was able to rescue molecular defects in the posterior region of the Raldh2(-/-) mutant heart, in a gene dosage-dependent manner.     

Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis

Heart formation requires the coordinated recruitment of multiple cardiac progenitor cell populations derived from both the first and second heart fields. In this study, we have ablated the Bmp receptor 1a and the Wnt effector beta-catenin in the developing heart of mice by using MesP1-cre, which acts in early mesoderm progenitors that contribute to both first and second heart fields. Remarkably, the entire cardiac crescent and later the primitive ventricle were absent in MesP1-cre; BmpR1a(lox/lox) mutants. Although myocardial progenitor markers such as Nkx2-5 and Isl1 and the differentiation marker MLC2a were detected in the small, remaining cardiac field in these mutants, the first heart field markers, eHand and Tbx-5, were not expressed. We conclude from these results that Bmp receptor signaling is crucial for the specification of the first heart field. In MesP1-cre; beta-catenin(lox/lox) mutants, cardiac crescent formation, as well as first heart field markers, were not affected, although cardiac looping and right ventricle formation were blocked. Expression of Isl1 and Bmp4 in second heart field progenitors was strongly reduced. In contrast, in a gain-of-function mutation of beta-catenin using MesP1-cre, we revealed an expansion of Isl1 and Bmp4 expressing cells, although the heart tube was not formed. We conclude from these results that Wnt/beta-catenin signaling regulates second heart-field development, and that a precise amount and/or timing of Wnt/beta-catenin signaling is required for proper heart tube formation and cardiac looping.     

Partial rescue of the Tbx1 mutant heart phenotype by Fgf8: Genetic evidence of impaired tissue response to Fgf8

Tbx1 is the candidate gene of DiGeorge syndrome and is required in humans and mice for the development of the cardiac outflow tract (OFT) and aortic arch arteries. Loss of function mutants present with reduced cell proliferation and premature differentiation of cardiac progenitor cells of the second heart field (SHF). Tbx1 regulates Fgf8 expression hence the hypothesis that the proliferation impairment may contribute to the heart phenotype of mutants. Here we show that forced Fgf8 expression modifies and partially rescues the OFT septation defects of Tbx1 mutants but only if there is some residual expression of Tbx1. This genetic experiment suggests that Tbx1, directly or indirectly, affects tissue response to Fgf8. Indeed, Tbx1^−/− mouse embryonic fibroblasts were unable to respond to Fgf8 added to the culture media and showed defective response of Erk1/2 and Rsk1. Our data suggest a coordinated pathway modulating Fgf8 ligand expression and tissue response to it in the SHF.     

Cardiac origin of smooth muscle cells in the inflow tract

Multipotent Isl1⁺ heart progenitors give rise to three major cardiovascular cell types: cardiac, smooth muscle, and endothelial cells, and play a pivotal role in lineage diversification during cardiogenesis. A critical question is pinpointing when this cardiac-vascular lineage decision is made, and how this plasticity serves to coordinate cardiac chamber and vessel growth. The posterior domain of the Isl1-positive second heart field contributes to the SLN-positive atrial myocardium and myocardial sleeves in the cardiac inflow tract, where myocardial and vascular smooth muscle layers form anatomical and functional continuity. Herein, using a new atrial specific SLN-Cre knockin mouse line, we report that bipotent Isl1⁺/SLN⁺ transient cell population contributes to cardiac as well as smooth muscle cells at the heart-vessel junction in cardiac inflow tract. The Isl1⁺/SLN⁺ cells are capable of giving rise to cardiac and smooth muscle cells until late gestational stages. These data suggest that the cardiac and smooth muscle cells in the cardiac inflow tract share a common developmental origin. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

Endogenous retinoic acid regulates cardiac progenitor differentiation

Retinoic acid (RA) has several established functions during cardiac development, including actions in the fetal epicardium required for myocardial growth. An open question is if retinoid effects are limited to growth factor stimulation pathway(s) or if additional actions on uncommitted progenitor/stem populations might drive cardiac differentiation. Here we report the dual effects of RA deficiency on cardiac growth factor signaling and progenitor/stem biology using the mouse retinaldehyde dehydrogenase 2 (Raldh2) knockout model. Although early heart defects in Raldh2^−/− embryos result from second-heart-field abnormalities, it is unclear whether this role is transient or whether RA has sustained effects on cardiac progenitors. To address this, we used transient maternal RA supplementation to overcome early Raldh2^−/− lethality. By embryonic day 11.5–14.5, Raldh2^−/− hearts exhibited reduced venticular compact layer outgrowth and altered coronary vessel development. Although reductions in Fgf2 and target pERK levels occurred, no alterations in Wnt/β-catenin expression were observed. Cell proliferation is increased in compact zone myocardium, whereas cardiomyocyte differentiation is reduced, alterations that suggest progenitor defects. We report that the fetal heart contains a reservoir of stem/progenitor cells, which can be isolated by their ability to efflux a fluorescent dye and that retinoid signaling acts on this fetal cardiac side population (SP). Raldh2^−/− hearts display increased SP cell numbers, with selective increases in expression of cardiac progenitor cell markers and reduced differentiation marker levels. Hence, although lack of RA signaling increases cardiac SP numbers, simultaneous reductions in Fgf signaling reduce cardiomyocyte differentiation, possibly accounting for long-term defects in myocardial growth.     

转录因子Tbx18及Wt1在小鼠心脏发育过程中的时空表达

目的:原位观察转录因子Tbx18及Wt1在胚胎心脏中的表达,及心肌细胞本身是否表达Tbx18及Wt1。方法:收集不同发育阶段小鼠胚胎(E)心脏,冰冻切片后,取不同区域的组织进行免疫荧光染色和DAPI染核,检测Tbx18、Wt1及Nkx2.5的表达。结果:小鼠前体心外膜及不同发育阶段的心外膜可明显表达Tbx18及Wt1蛋白,但未检测到心脏转录因子Nkx2.5的表达。从E10.5~至少E14.5d,Tbx18蛋白明显表达于不同区域的心脏组织中。除E14.5 d少许表达Tbx18的细胞不表达Nkx2.5外,这些表达Tbx18的细胞还同时表达Nkx2.5。从E12.5~至少14.5 d,Wt1表达于不同区域心脏组织中,但这些表达Wt1的细胞都不表达NKx2.5。结论:从E10.5~至少14.5 d,Tbx18表达于小鼠心肌细胞中;从E12.5d~至少E14.5d,Wt1表达于小鼠心脏组织中,但不表达于心肌细胞中。     

Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction

Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5'-azacytidine, in part depending on Bmpr1a, a receptor for bone morphogenetic proteins. Given intravenously after ischemia/reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre/Lox donor/recipient pair (alphaMHC-Cre/R26R), differentiation was shown to occur roughly equally, with and without fusion to host cells.