Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway

Abstract

Distinct progenitor cell populations exist in cardiac mesoderm important for patterning of the heart. During heart tube formation in mouse, Tbx5 is expressed in progenitors located more laterally, whereas Isl1 and Fgf8 are expressed in progenitors located more medially. Signals that drive mesodermal progenitors into various cardiac lineages include Fgf8, which functions to induce Isl1. Studies in chick and zebrafish have shown that retinoic acid restricts the number of cardiac progenitors, but its role in mammalian cardiac development is unclear. Here, we demonstrate that Raldh2(-/-) mouse embryos lacking retinoic acid signaling exhibit a posterior expansion of the cardiac Fgf8 expression domain as well as an expansion of Isl1 expression into mesoderm lying posterior to the cardiac field. We provide evidence that retinoic acid acts specifically in the posterior-medial region of the cardiac field to establish the heart posterior boundary potentially by reducing Fgf8 expression which restricts the Isl1 domain.

Fig. 1

RA rescue targets the posterior-medial region of the heart field during heart tube formation. (A–B) Detection of Raldh2 mRNA in E9.5 wild-type (WT) and Raldh2^−/− embryos; mutant lacks Raldh2 mRNA and exhibits a medial distended cardiac cavity. (C) RA activity in 6-somite wild-type and Raldh2^−/− embryos carrying RARE-lacZ RA-reporter transgene following rescue by maternal dietary RA administration (ventral view); mutant exhibits RA activity only in the neuroectoderm and posterior-medial region of the heart field. (D–E) Serial transverse sections through the RA-rescued Raldh2^−/− embryo indicated in panel C. (F) RA activity in 1-somite Raldh2^−/− embryo rescued by maternal dietary RA administration (ventral view); RA activity is observed in the posterior region of the heart field and in the posterior neuroectoderm. (G) Transverse section through the RA-rescued Raldh2^−/− embryo indicated in panel F. atr, atrial progenitor; cc, cardiac crescent; h, heart; n, neuroectoderm; s, somite; sm, splanchnic mesoderm (component of lateral plate mesoderm); sv, sinus venosa (inflow tract); ven, ventricular progenitor.

Fig. 2

Isl1⁺ cardiac domain expands posteriorly after loss of RA. Isl1 mRNA in wild-type and Raldh2^−/− embryos at (A–B) 3-somites, ventral view. and at (C–D) 5-somites, ventral view. Brackets indicate a marked increase in Isl1 mRNA in the posterior cardiac field of mutants. (E–F) Spry2 mRNA in 3-somite wild-type and Raldh2^−/− embryos (ventral view) demonstrating a significant increase in posterior cardiac expression in the mutant (brackets); transverse sections shown in panels I and J. (G–H) Isl1 mRNA in wild-type and Raldh2^−/− embryos at 6-somites (ventral view) with brackets pointing out posterior expansion of Isl1 mRNA in mutant; transverse sections shown in panels K–N. sm, splanchnic mesoderm.

Fig. 3

Loss of RA reduces the size of the Tbx5⁺ cardiac domain. (A–B) Tbx5 mRNA in 3-somite wild-type and Raldh2^−/− embryos (lateral view); brackets point out reduced Tbx5 mRNA in posterior region of cardiac crescent in mutant. (C–D) Ventral view of 3-somite embryos shown in panels A and B. (E) Double-labeling of Tbx5 mRNA (anterior) and Raldh2 mRNA (posterior) in 6-somite wild-type and Raldh2^−/− embryos (ventral view); mutant lacks Raldh2 mRNA; brackets identify reduction in posterior-lateral Tbx5 domain in mutant. (F) Detection of Raldh2 mRNA in a 6-somite wild-type embryo (compare to panel E). cc, cardiac crescent; s, somite; sm, splanchnic mesoderm.

Fig. 4

Posterior expansion of cardiac Fgf8 expression following loss of RA signaling. (A–B) Detection of Fgf8 mRNA in 1-somite wild-type and Raldh2^−/− embryos (lateral view); arrows point out posterior expansion of the cardiac domain and anterior expansion of the primitive streak domain in the mutant. (C) Ventral view of 1-somite embryos in panels A and B; note posterior expansion of Fgf8 mRNA in the heart field. (D–E) Detection of Fgf8 mRNA in 5-somite wild-type and Raldh2^−/−embryos; arrows show posterior expansion in heart field. (F–G) Transverse sections of 1-somite wild-type and Raldh2^−/− embryos through posterior region of Fgf8-positive cardiac domain (just below the label ‘heart’ in embryos shown in panel C). h, heart; n, neural tube; no, node; ps, primitive streak; s, somite; sm, splanchnic mesoderm.

Fig. 5

RA treatment represses Fgf8 and Isl1 expression. (A–C) Fgf8 mRNA in 1-somite embryos, and (D–F) Isl1 mRNA in 6-somite embryos; all embryos are displayed from a ventral view to observe the heart field. The arrows indicate posterior expansion of Fgf8 or Isl1 expression in untreated Raldh2^−/− embryos relative to wild-type, but this posterior expansion is prevented in Raldh2^−/−embryos treated with maternal dietary RA supplementation (res −/−).