Retinoic acid deficiency alters second heart field formation

Results

To assess retinoid activity in the WT cardiac field, we used mice harboring the RARE-hsp68-lacZ transgene, a sensitive reporter for endogenous RA activity (17). This transgene has been shown to be expressed in the posterior region of the early heart (8), and we confirmed that expression was localized to two dorsal–medial streams of the forming heart tube (Fig. 1A, arrowheads), a domain absent in Raldh2^−/− mutants (data not shown). At the five-somite stage, Raldh2 and Tbx5 expression domains converged to overlap in the most posterior cardiac precursors in the splanchnic mesoderm (Fig. 1 B and C). Similar domains of the RA-responsive Hoxa1 homeobox gene (18) were present adjacent to the cardiac field in WT embryos at the five- to eight-somite stages (Fig. 1 D, E, and G), but these domains were absent in Raldh2^−/− embryos (Fig. 1 F and H).

We crossed Raldh2^+/− mice with the Mlc1v-nlacZ-24 line, in which a transgene integration into the Fgf10 locus leads to transgene expression in the cardiac progenitors located in the anterior heart field and, because of perduration of β-galactosidase (β-gal), activity allows for marking the corresponding myocardial derivatives (16). Although the early expression of this reporter transgene did not show a major difference between WT and Raldh2^−/− mutants at the one- to two-somite stages (Fig. 2 A and B), from the five-somite stage, its expression was clearly altered in mutants. First, the labeled cells were dramatically expanded toward posterior regions (compare Fig. 2 C and D, arrowheads), an alteration that was accentuated at the seven-somite stage (Fig. 2 E–J′). Thus, in Raldh2^−/− mutants the X-gal staining was present in the lateral mesoderm well beyond the level of the developing heart tube (compare Fig. 2 I′ with J′, arrowheads). Second, the closure of the dorsal mesocardium was defective, because the midline gap in X-gal staining became progressively wider in the Raldh2^−/− mutants (Fig. 2 D, F, and J, brackets). To examine the activation of the Mlc1v-nlacZ-24 transgene in the lateral mesoderm lying posterior to the heart tube, explants from this region were dissected at the zero- to two-somite stage (a stage at which the transgene is not activated posteriorly in Raldh2^−/− embryos; Fig. 2 A and B) and cultured for 36 h. Explants from Raldh2^−/− mutants were X-gal-positive after the in vitro culture (data not shown), indicating that the expanded Mlc1v-nlacZ-24 pattern observed in whole embryos does not result from an abnormal migration of anterior cells. At later stages [embryonic day (E)9.0], X-gal activity in Mlc1v-nlacZ-24;Raldh2^−/− mutants was spatially reduced compared with control embryos (Fig. 2 K and L). The staining adjacent to the WT second and third pharyngeal arches (Fig. 2K, arrowheads) was absent in mutants, although expression was detected in the anterior portion of the “unlooped” heart tube (Fig. 2 K and L).

We examined expression of other anterior markers of the SHF such as Fgf8 and Tbx1. Fgf8 expression was more intense and caudally expanded in Raldh2^−/− mutants, from the three- to four-somite stage onward (Fig. 3 A–D). As for the Mlc1v-nlacZ-24 transgene, Fgf8 expression was detected in the posterior lateral mesoderm of the Raldh2^−/− mutant embryos at the eight-somite stage (Fig. 3 C′ and D′; the greater expansion of the Mlc1v-nlacZ-24 transgene may be attributable to β-gal stability). The T-box gene Tbx1, frequently deleted in human 22q11 deletion DiGeorge syndrome (reviewed in ref. 15), appears to be genetically upstream of Fgf8 (19–22). WT embryos exhibited pronounced Tbx1 expression in the SHF region caudal–medial to Mlc1v-nlacZ-24 reporter staining (Fig. 3 E and E′), whereas Tbx1 expression appeared more intense and expanded in range in Raldh2^−/− mutants starting at the three- to four-somite stage (Fig. 3 F and F′ and data not shown). We also examined expression of a pan-SHF progenitor cell marker, Isl1 (10). Isl1-expressing cells in the splanchnic mesoderm give rise to cardiomyocytes of the OFT, the right ventricle, atria, and inflow region of the heart (10). Isl1 is also expressed in ventral and lateral foregut endoderm (see Fig. 3G′). Caudal expansion of Isl1 expression was observed in Raldh2^−/− mutants (Fig. 3 G and H), but the labeled cells did not extend into the heart tube where cardiomyocytes are present (Fig. 3 G′ and H′). Altogether, these observations suggest that endogenous RA may control the antero-posterior location of cardiac progenitors of the SHF within the splanchnic mesoderm.

Excess RA levels can often affect the same RA-regulated signaling pathways as RA deficiency but in an opposite manner. This was found to be the case when a single dose of RA was administered to WT mice at late gastrulation (E7.5). These treatments resulted in marked reductions of endogenous Fgf8 and Isl1 transcripts and of Mlc1v-nlacZ-24 reporter transgene staining [supporting information (SI) Fig. 6].

We then examined Raldh2^−/− mutants for endogenous expression of Bmp2, -4, -6 and -7, which encode TGFβ family members influencing myocardial differentiation (reviewed in ref. 23). We found Bmp6, Bmp7 (SI Fig. 7 A–D), and Bmp2 (see Fig. 5 F and H) levels to be reduced in the inflow region of the mutants. Interestingly, at later stages (E9.0), Tgfβ2 expression was markedly increased at the venous pole (septum transversum) of the Raldh2^−/− unlooped heart (SI Fig. 7 E and F). A similar hyperactivation of TGFβ signaling is observed in foregut tissues of Raldh2^−/− mutants at E8.5 and has been implicated in the lack of lung induction (24).

The reductions in levels of Mlc1v-nlacZ-24 transgene expression (an Fgf10 enhancer trap) in the pharyngeal mesoderm of Raldh2^−/− mutants at E9 (Fig. 2 K and L) suggest that FGF signaling may be affected in the SHF. To further investigate this pathway, we performed immunolocalization of p-ERK1/2, effectors of MAP signaling whose phosphorylation correlates with FGF activity in the cardiac region (25). p-ERK1/2 levels were found to be lower in the SHF at the 10-somite stage in Raldh2^−/− embryos (SI Fig. 7 G and H). Consistent with the decrease of p-ERK levels, the expression of other FGF downstream targets (Sprouty1 and -2 and Mkp3; SI Fig. 7 I–J′ and data not shown) was also reduced in the pharyngeal region and splanchnic mesoderm of mutants.

To examine myocardial subdomains in the RA-deficient hearts, we crossed Raldh2^+/− mutants with additional, region-specific reporter transgenes. The y96-Myf5-nlacZ-16 transgene displays a domain of expression in the WT OFT myocardium beginning at the 12- to 14-somite stage (26), which was absent in Raldh2^−/− mutants (Fig. 4 A and B). The absence or reduction of OFT myocardium in mutants was confirmed by the Mlc3f-nlacZ-9 pattern (Fig. 4 C and D). This transgene is known to be silenced in WT OFT myocardium (12) (Fig. 4C, bracket). In mutants, most if not all of the unlooped heart tube contained Mlc3f-nlacZ-9-labeled cells (Fig. 4D). These observations indicate that the deployment of SHF cells to the OFT was highly compromised in Raldh2^−/− mutant embryos. In contrast, the activity of the Mlc3f-nlacZ-2E transgene, normally expressed in left but not right ventricular myocardium (Fig. 4 E and G), was well detected in the Raldh2^−/− heart tube (compare Fig. 4 E and G with F and H) (12). These results strongly suggest that FHF contribution is not affected under RA deficiency. To estimate the myocardial cell numbers and the presence of differentiating cardiomyocytes in the Raldh2^−/− mutant hearts, we counted the number of β-gal-positive and α-actinin-positive cells on serial sections of WT and Raldh2^−/− embryos from the Mlc3f-nlacZ-9 line (SI Fig. 8). At E9.5, we estimated a deficiency of ≈30%, both in the overall number of β-gal-positive cells and of differentiating (α-actinin-positive) cardiomyocytes in Raldh2^−/− mutants, which may correspond to the defects observed in the outflow and inflow regions.

To further explore the differentiation potential of Raldh2^−/− cardiomyocytes, explants of SHF isolated from three- to five-somite stage WT and Raldh2^−/− embryos carrying the Mlc1v-nlacZ-24 transgene were cultured for 48 h and X-gal-stained to identify SHF cells and derivatives (12, 16). Under these established culture conditions, WT SHF explants consistently underwent cardiac differentiation, characterized by the presence of beating cells and expression of myosin heavy chain (MF20), quite similar to heart explants cultured from the same embryos (SI Table 1; also see ref. 12). Raldh2^−/− mutant SHF explants, however, never differentiated or formed beating cardiomyocytes, in contrast to their heart, which was still beating under the same culture conditions (SI Table 1). This suggested that the Raldh2^−/− mutant SHF was impaired in its ability to contribute to differentiated cardiomyocytes, potentially because of reduced bone morphogenetic protein (BMP) signaling.

The intriguing finding that both Raldh2 deficiency and RA receptor antagonism in zebrafish embryos resulted in the production of excess myocardial progenitors, marked by up-regulated Nkx2.5 expression during gastrulation (5), led us to investigate potential mechanisms for cross-regulation between the retinoid and Nkx2.5 transcriptional pathways. One avenue to examine such interactions is to generate compound mutants. Insertion of a Cre transgene in the Nkx2.5 locus (27) disrupts expression of this gene, so that when mated to homozygosity, the resulting embryos phenocopy previously reported Nkx2.5^−/− mutants (27–29). We crossed Nkx2.5-Cre mutants with Raldh2^+/− mice and analyzed compound mutants at E8.5 for Tbx5 expression as a marker for the posterior portion of the heart, which is destined to become the sinus venosa and atria (30). As expected, the domain of Tbx5 expression was reduced in Raldh2^−/− embryos (compare Fig. 5A with C), correlating with an abnormal morphogenesis of the inflow region (4). Interestingly, Nkx2.5 haploinsufficiency partially rescued Tbx5 expression in Raldh2^−/− embryos (Fig. 5D). Complete Nkx2.5 deficiency further expanded Tbx5 expression in Raldh2^−/−; Nkx2.5^−/− compound mutants (Fig. 5E). Prall et al. (31) have demonstrated that Nkx2.5 deficiency up-regulates Bmp2 expression, which may result in increased myocardial differentiation. We likewise observed expanded Bmp2 expression in our Nkx2.5^−/− mutants (Fig. 5G). Although Bmp2 levels were reduced in Raldh2^−/− mutants (Fig. 5H), we found that these levels were progressively normalized upon removal of functional Nkx2.5 alleles (Fig. 5 I and J). Additionally, SHF expression, as assayed by Isl1 expression, appeared essentially unaltered in Raldh2^−/− mutants under Nkx2.5 deficiency (data not shown).

By performing a VISTA-based promoter search for transcriptional binding sites in evolutionary conserved regions, we uncovered an upstream regulatory region of the Nkx2.5 gene containing five conserved RA-response elements (RAREs) of the DR5 type. A lacZ transgenic line was generated by using a BAC fragment containing the region located from −14.8 to −7.5 kb upstream of the Nkx2.5 transcriptional start site, which included the RAREs. This fragment was sufficient to drive lacZ reporter expression in the FHF (SI Fig. 9 A–C), similar to endogenous Nkx2.5 expression (28). Whereas the early domain of endogenous Nkx2.5 (data not shown) and Nkx2.5 transgene expression in the cardiac crescent was not altered in Raldh2^−/− mutants (SI Fig. 9 D–F), endogenous Nkx2.5 was expanded in the SHF of mutants at E8.5 (SI Fig. 9 G and H, arrowheads). However, transgene expression was reduced in the OFT and the right ventricle of RA-supplemented Raldh2^−/− embryos at E10.5 (SI Fig. 9 I and J). These observations suggest that Nkx2.5 regulation is not RA-dependent in the FHF and at early stages in the SHF, although eventually it is affected when RA levels are decreased at later stages of development.

Discussion

Originally RA was proposed to regulate the anterior–posterior specification of cardiac progenitors within the FHF. Indeed, posterior cells of the early FHF are encircled by Raldh2-expressing cells in the lateral mesoderm. Blocking RA signaling in chick embryos inhibited atrial differentiation and altered the cardiac fate map, so that ventricular progenitors extended outside of their normal domains (7). Keegan et al. (5) further have found that RA deficiency results in an expansion of cardiac progenitors during gastrulation in zebrafish, via a direct fate transformation.

Our results, although partly consistent with previous findings, now show that RA regulates SHF organization within the precise time frame in which RA deficiency affects cardiac formation in avian and mammalian models. We also observed an overlap between the region of RA signaling and the caudal limit of SHF marker gene expression, correlating with an anterior domain of Hoxa1 expression in WT embryos, a domain that is absent in Raldh2 mutants. Because restoring Hoxa1 expression in RA-deficient amphioxus embryos can rescue patterning defects (32), we postulate that the lack of Hoxa1 expression in the cardiac field may result in cardiac perturbations found in Raldh2 mutants (4). We found that the earliest events of cardiac RA deficiency were the caudal expansions of SHF gene expression, occurring at a stage when cardiac defects become irreversible in an avian vitamin A-deficiency model (33). We propose that these initial molecular alterations in SHF expression are why RA-deficient heart formation cannot be restored after this time. Additional reductions in the right ventricle and absence of OFT because of impaired SHF integration in the forming heart tube probably contributes to abnormal cardiac looping also found in other SHF mutants such as Isl1, Mef2c, and Nkx2.5 (10, 31, 34).

Some genetic cross-talk exists between Nkx2.5 and RA signaling, because we show here that reducing Nkx2.5 gene dosage can rescue aspects of the Raldh2^−/− cardiac phenotype, including Tbx5 expression. Nkx2.5^−/− mutants have an apparent expansion of the FHF contribution to the left ventricle (31). Hence it may have been expected that Nkx2.5 loss of function would have worsened the Raldh2^−/− cardiac phenotype. More likely it is the feedback repression that Nkx2.5 normally imposes on Bmp2/Smad signaling (31), which when relieved may improve the RA deficiency phenotype. Transplantation of WT foregut endoderm, or exogenous supply of BMP2 and Gata4, can restore heart morphology in vitamin A-deficient quail embryos (35, 36). Although Bmp2 expression indeed appears up-regulated in Nkx2.5;Raldh2 compound mutants, further genetic strategies are required to clarify the precise manner by which these two signaling pathways interact.

The importance of Fgf8 in SHF signaling has been noted (19, 20, 22, 37, 38). Our study indicates that a primary mechanism by which RA regulates SHF dynamics may be by modulating FGF signaling. We show that endogenous RA is required to prevent Fgf expression in the posterior lateral mesoderm (including in the posterior region of the SHF). Not only did RA deficiency lead to expanded Fgf8 and Fgf10 (Mlc1v-nlacZ-24-enhancer trap) expression, this expansion occurred at approximately the five-somite stage, when cardiac defects become irreversible in the quail vitamin A-deficiency model (33). Conversely, we saw that excess exogenous RA reduces SHF Fgf8 levels, similar to what has been observed when endogenous RA is increased by blocking the activity of its metabolizing CYP26 enzymes (39).

One puzzling finding in Raldh2 mutants was that despite the expansion of the SHF at early stages of development, eventually the contribution of SHF cells to the heart tube was compromised. Explant studies demonstrate that mutant SHF cells are impaired in their ability to differentiate and to form beating cardiomyocytes. Several explanations may account for these findings. Reduction in BMP signaling, found in Raldh2 mutants, may lead to the absence of bona fide cardiac induction. Accordingly, FGFs can only elicit cardiomyogenic effects in regions where BMP signaling is also present (40). Alternately, the expanded early FGF8 signaling may hinder the capacity of the prospective myocardial SHF cells to develop and correctly function, similar to what is observed at slightly later stages when excess FGF8 resulting from cardiac neural crest ablation disrupts myocardial function (37, 41). Moreover, altering RA-Fgf8 regulatory interactions may produce adverse consequences. In prostate cancer cells, RA activation of the Fgf8 promoter produces a switch of splice isoforms, so that a less mitotically active isoform (Fgf8a) accumulates (42). Because both canonical and novel RA-response elements in the Fgf8 promoter can be activated by unliganded RARα in COS cells (43), an absence of RA may produce an accumulation of the more proliferative Fgf8b product. Hypothetically, the absence of RAR ligand in Raldh2^−/− mutants may favor Fgf8b expression, hindering early SHF progenitor differentiation by its increased mitotic activity.

In conclusion, mutual inhibition between FGF and RA pathways may control cardiac stem cell dynamics and cardiomyocyte differentiation, similar to what has been described during caudal neuronal stem cell production and neural tube differentiation (reviewed in ref. 44). In this tail bud of chick embryos at early somite stages, Fgf8 expression marks the caudal source of neuronal stem cells, which under RA deficiency is expanded. The ability of neural progenitors to differentiate, however, is impaired when RAR signaling is blocked (44). Similarly we observed that cardiac stem cell populations, marked by Isl1 expression (45), were expanded under RA deficiency, whereas SHF cardiomyocyte differentiation was impaired. Further understanding of how RA regulates cardiac progenitors and growth factor pathways in the early embryo may engender novel ideas about how to regulate cardiac stem cell dynamics in humans.

Materials and Methods

Supplementary Material