Retinoic acid deficiency alters second heart field formation

Abstract

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.

Fig. 1.

Retinoid signaling in the early mouse heart. (A–D) Ventral views of WT embryos at the five- to six-somite stage (5–6s). (A′–D′) Cryosections of the embryos shown in A–D, with dotted lines indicating planes of sections. (A and A′) WT RARE-hsp68-lacZ transgenic embryo at six-somite stage exhibits a domain of retinoid activity in the splanchnic mesoderm (spm) adjacent to the forming heart tube (A, arrowheads). (B and B′) Whole-mount ISH of Tbx5 in a five-somite-stage WT embryo shows robust expression in the posterior most heart portion. (C and C′) Double ISH of Tbx5 and Raldh2 in a five-somite-stage WT embryo reveals complementary, although partly overlapping, expression domains in the splanchnic mesoderm. (D and D′) Double ISH of Tbx5 and Hoxa1 in a five-somite-stage WT embryo shows adjacent expression domains in splanchnic mesoderm. Whole-mount ISH of Hoxa1 in WT (E and G) and Raldh2^−/− (F and H) embryos indicates that RA is required to induce Hoxa1 expression adjacent to the cardiac field (E and F) and caudal pharyngeal field (G and H). The arrowheads (E) indicate expression nearest to the cardiac field. ba, first branchial arch; fg, foregut; h, forming heart tube; lv, left ventricle; sm, somatic mesoderm; spm, splanchnic mesoderm.

Fig. 2.

Retinoid deficiency causes SHF defects. WT and Raldh2^−/− Mlc1V-nlacZ-24 transgenic embryos were analyzed by X-gal staining (A–F, ventral views; G, H, K, and L, right-side views; I–J′, transverse sections). At the one- to two-somite stage, transgene expression is unaltered in mutants (A and B). At the five-somite (C and D) and seven-somite (E and F) stages, red arrowheads point to the caudal limit of expression in the SHF, which is expanded in Raldh2^−/− mutant embryos (D and F). Note the abnormally large gap in midline staining in mutants (brackets). (G and H) Right-side views of embryos shown in E and F. The arrows indicate planes of sections in I–J′. (I and J) Transverse sections reveal β-gal⁺ cells in splanchnic epithelium (white arrowheads) adjacent to foregut endoderm, in somatic mesoderm (black arrowheads), and in heart tube myocardium. Note that dorsal mesocardium is not closed in the Raldh2^−/− embryo (J, bracket). (I′ and J′) Expansion of Mlc1v-nlacZ-24 transgene expression is seen in posterior lateral mesoderm (arrowheads) of the Raldh2^−/− mutant (J′). (K and L) Right-side views of E9.0 WT and Raldh2^−/− embryos, showing details of the heart and adjacent pharyngeal region. The arrowheads point to pharyngeal staining, which is almost undetectable in the mutant. fl, forelimb; ht, heart tube; ot, OFT; rv, right ventricle.

Fig. 3.

Caudal expansion of the SHF in RA-deficient mutants. Whole-mount ISH analysis of Fgf8 (A–D′), Tbx1 combined with X-gal staining for Mlc1v-nlacZ-24 (E–F″) and Isl1 (G–H′). The arrows or dotted lines in C–H indicate planes of sections shown in C′–H′. (A–D) Brackets indicate endogenous Fgf8 expression in the anterior part of the SHF, which is expanded in Raldh2^−/− mutants (B and D). (D′) A transverse section of the embryo shown in D reveals Fgf8 expression in the lateral mesoderm. (E and F) Ventral of WT and Raldh2^−/− embryos at the three- to four-somite stage showing a caudal expansion of Mlc1v-nlacZ-24 transgene expression and Tbx1. (E′–F″) Transverse sections further illustrate the caudal expansion of Mlc1v-nlacZ-24 transgene activity and Tbx1 transcripts. (G and H) Right-side views of WT and Raldh2^−/− embryos at the 10-somite stage showing an expansion of the pan-SHF marker Isl1 under RA deficiency (H). (G′ and H′) Transverse sections show Isl1 expression in the splanchnic mesoderm (the SHF) and the ventral foregut endoderm of both embryos. Of note, there is no ectopic expression of Isl1 transcripts in the myocardium of the mutant heart (compare G′ with H′). cm, cranial mesoderm; dm, dorsal mesocardium; lm, lateral mesoderm.

Fig. 4.

Transgenic reporter lines reveal OFT defects and normal FHF contribution in Raldh2^−/− embryos. (A and B) Right-side views of X-gal stained E9 WT and Raldh2^−/− embryos, showing absence of y96-Myf5-nlacZ-16 transgene expression, a marker of the OFT (arrowheads), in the mutant. (C and D) In Mlc3f-nlacZ-9 hearts at the 14-somite stage, X-gal staining distinguishes the OFT lacking transgene activity (C, bracket) and the right ventricle. β-Gal activity is also detected in the left ventricle. The nonexpressing domain (presumptive OFT) is absent under RA deficiency (D). The white lines indicate planes of sections in C′ and D′. (E and F) At the eight-somite stage, Mlc3f-nlacZ-2E transgene expression marks the presumptive right atria and left ventricle in both WT (E) and Raldh2^−/− mutants (F). (G and H) Ventral views of X-gal stained Mlc3f-nlacZ-2E WT and Raldh2^−/− embryos at the 16-somite stage showing the distinction between right (arrowheads) and left ventricles. White lines indicate planes of sections in G′ and H′. ra, right atria.

Fig. 5.

Genetic interaction between the retinoid and Nkx2.5 pathways. Sinoatrial Tbx5 expression (A, WT embryo) is reduced in Raldh2^−/− mutants (C). Inactivation of one Nkx2.5 allele results in an expanded Tbx5 domain in Raldh2^−/− mutants (D), similar to Nkx2.5^−/− mutants (B) and Nkx2.5^−/−;Raldh2^−/− double mutants (E). Bmp2 expression (F, WT embryo) is increased in Nkx2.5^−/− mutants (G) and moderately reduced in Raldh2^−/− mutants (H). Inactivation of one (I) or both (J) Nkx2.5 allele(s) progressively increases Bmp2 levels in Raldh2^−/− mutants. All embryos are at the 8- to 10-somite stage. Profile views are shown.