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Abstract Notch signaling has been shown to mediate a wide array of cell fate decisions during development. While previous work has demonstrated that Notch signaling plays an important role in regulating cardiac differentiation and morphogenesis, an earlier role during cardiac field formation has not yet been fully characterized. Previously, our lab demonstrated that perturbations in Notch signaling beginning at the onset of gastrulation affect the subdivision of germ layers. However due to the potential additive effects of misregulating Notch signaling over multiple stages of development, it was not possible to distinguish a specific role for this pathway during heart field specification. Here, we developed an innovative approach that takes advantage of temporally inducible constructs to isolate our manipulations to specific windows of development. In particular, we focused our studies on some of the earliest stages of cardiogenesis when heart field specification occurs. Our findings demonstrate a novel role for Notch signaling during the prepatterning of the cardiac mesoderm. Specifically, once relieved of aberrantly activated Notch signaling following gastrulation, cardiac precursors retain the ability to express markers of the cardiac field. Conversely, downregulating Notch signaling in cells fated to become hearttissue results in the induction of cardiac field genes in gastrula embryos. Finally, we provide evidence suggesting that this new role for Notch signaling is mediated at least in part via the Notch effector protein, Esr9 and the transcription factor GATA4. Taken together, these findings provide strong evidence for a novel role for Notch signaling in regulating the timing of heart field specification during early cardiogenesis.
Fig.1. The expression of the Notch receptor xotch and ligand delta1 in the precardiac mesoderm during early cardiogenesis. Embryos were examined by in situ hybridization analysis for the expression of xotch and delta1 in gastrula and early neurula embryos. Gastrula-stage whole embryos are viewed from the vegetal pole with the dorsal side located at the top of panels (A–D). The plane of the midsagittal sections of sibling embryos (A’, B’, C’, D’) is denoted by a dashed line in adjacent panels. Midsagittal sections are viewed with the animal pole at the top. Early neurula-stage embryos are dorsal views with anterior toward the top in (E, F), and anterior views with dorsal to the top in (E’, F’). In situ hybridization analysis reveals expression of the Notch receptor (xotch) and ligand (delta1) at the onset of gastrulation in the presumptive mesoderm of the marginal zone that narrows toward the dorsal side of embryos and is retained in cells migrating along the blastocoel roof (A–D’; arrow heads denote location of migrating xotch and delta1 positive cells). Once embryos have reached early neurula stages, the expression of both xotch and delta1 has become more restricted and is now expressed primarily in neural cell types (E–F’). Anterior views show that xotch and delta1 are expressed in the neural plate, but expression does not extend to the more ventrally located cardiac field at these stages (E’, F’; full arrows).
Fig.2. The HES genes esr9 and esr10 are Notch-responsive in marginal zone mesoderm during gastrulation. All images are gastrula stage whole embryos viewed from the vegetal pole. (A, D) In situ hybridization analysis of uninjected embryos shows that the expression of the putative downstream Notch effector genes, esr9 and esr10, are expressed in the marginal zone in a ring pattern coinciding with delta, and xotch expression (Fig. 1). Embryos were co-injected with a lineage label and either GR-Su(H)VP16 to upregulate Notch signaling (B, E) or GR-Su(H)DBM to downregulate the Notch pathway (C, F). Constructs were induced at the onset of gastrulation by the addition of dexamethasone to the culture medium and collected 1 h later. Arrowheads indicate the location of the progeny of the injected cells as detected by the presence of magenta-gal-positive nuclei. (B, E) An expansion of esr9 and esr10 expression resulted when Notch signaling was upregulated (esr9 78% of embryos, n = 120; esr10 82% of embryos, n = 82) and decreased expression was observed when Notch signaling was downregulated (esr9 68% of embryos, n = 96; esr10 75% of embryos, n = 52). On average, 21% or less of both uninjected-control embryos and injected embryos not exposed to hormone were observed to contain alterations in normal esr9 and esr10 expression (normal esr9 expression was observed in: 91% of embryos injected with GR-Su(H)VP16/no hormone, n = 104; 85% of embryos injected with GR-Su(H)DBM/no hormone, n = 128; normal esr10 expression was observed in: 91% of embryos injected with GR-Su(H)VP16/no hormone, n = 57; 79% of embryos injected with GR-Su(H)DBM/no hormone, n = 91). Percents represent the number of embryos with the phenotype described resulting from three replicate experiments conducted on different days.
Fig.3. Co-expression and co-induction of GR-Su(H)VP16 and ER-Su(H)DBM rescues normal gene expression. Embryos were co-injected with GR-Su(H)VP16 and/or ER-Su(H)DBM and either dexamethasone (Dex), 17β-estradiol (E2), or both hormones were added to the culture medium at stage 10, when heart field specification begins. Embryos were examined for nkx2.5 expression via in situ hybridization at tailbud stages. The percents on the graph summarizing these data represent the number of embryos from three experiments observed to contain an aberrant molecular phenotype (increase or decrease) on the injected side of the heart field as compared to normal nkx2.5 expression on the uninjected side of the same embryo (n = 50–100 animals for each treatment group). (A) When GR-Su(H)VP16 is injected alone and its expression is induced with dexamethasone to upregulate Notch signaling, nkx2.5 expression is dramatically decreased (treatments: Dex10 82% of embryos, n = 91; no hormone control 5% of embryos, n = 97). (B) Conversely, when ER-Su(H)DBM is injected alone and its expression is induced by 17β-estradiol to suppress Notch signaling, an increase in nkx2.5 gene expression is observed (treatments: E210 75% of embryos, n = 60; no hormone control 17% of embryos, n = 76). (C) Co-injection of GR-Su(H)VP16 and ER-Su(H)DBM plus the addition of only one of the two hormones, resulted in similar molecular phenotypes to those observed in embryos injected with one of the two Notch constructs (compare to A, B). As before, upregulated Notch signaling decreased nkx2.5 expression in the heart field (63% of embryos, n = 54) and downregulation of Notch expanded nkx2.5 expression (68% of embryos, n = 63). However, when the two co-injected Notch constructs were simultaneously induced via addition of both hormones (Dex10 and E210) to the culture medium, a rescue of the individual mutant phenotypes was observed (nkx2.5 normally expressed in 66% of embryos, n = 80). (D) Representative images of embryos co-injected with GR-Su(H)VP16 and ER-Su(H)DBM and either dexamethasone (Dex), 17β-estradiol (E2), or both hormones were added to the culture medium at stage 10. Ablated nkx2.5 expression in the Dex(10) treatment is restored in the dual-hormone treatment. Arrows indicate injected side of embryos. White line marks midline of ventral views of tailbud stage embryos.
Fig.5. The reduction of renal gene expression resulting from upregulated Notch signaling during kidney field patterning can be rescued. Embryos were co-injected with GR-Su(H)VP16 and/or ER-Su(H)DBM and inducing hormones were added at the stages indicated. Embryos were examined for the expression of renal markers lim-1, evi1, and pax8 via in situ hybridization at tailbud stages. The percents on the graph summarizing these data represent the proportion of embryos from two experiments observed to contain an aberrant molecular phenotype (increase or decrease) on the injected side of the embryo compared to the uninjected side (n = 16–27 embryos for each treatment group). (A) When GR-Su(H)VP16 was induced to upregulate Notch signaling at stage 10, renal marker expression was reduced (lim1 75% of embryos, n = 16; evi1 91% of embryos, n = 23; pax8 74% of embryos, n = 23). Conversely, when ER-Su(H)DBM was induced by 17β-estradiol to suppress Notch signaling, an increase in renal gene expression was observed (lim1 71% of embryos, n = 21; evi1 67% of embryos, n = 21; pax8 72% of embryos, n = 18). If GR-Su(H)VP16 was induced at stage 10 and ER-Su(H)DBM was induced at stage 14, normal renal marker expression was observed (lim1 80% of embryos, n = 20; evi1 78% of embryos, n = 23; pax8 71% of embryos, n = 21). (B–G) Representative images of embryos co-injected with GR-Su(H)VP16 and ER-Su(H)DBM and dexamethasone to upregulate Notch signaling (B–C), 17β-estradiol to downregulate Notch signaling (D–E), or both hormones to upregulate and then moderate Notch signaling (F–G) were added to the culture medium and examined for pax8 expression. Images are lateral views with anterior facing outwards. Arrows indicate the observed molecular phenotype of decreased (C), increased (E), or normal (G) pax8 expression.
Fig.6. Suppression of Notch signaling during gastrulation results in the robust expression of cardiac field genes. Gastrula 11–11.5 stage embryos are oriented laterally, vegetal to the left. (A, C) During gastrulation, cardiac field marker transcripts nkx2.5 and tbx5 are not detected by in situ hybridization analysis in uninjected embryos. (B, D) One blastomere of cleavage-stage embryos was injected with mRNAs encoding GR-Su(H)DBM plus a lineage marker. At the onset of gastrulation (stg. 10) constructs were induced via the addition of hormone. When Notch signaling was downregulated during heart field specification, the expression of both nkx2.5 (B; 76% of embryos, n = 117) and tbx5 (D; 79% of embryos, n = 86) robustly expressed in the migrating cardiac precursor cell population, whereas expression in uninjected embryos is difficult to detect via in situ hybridization at these stages (A, C). Black arrows denote the injected side of embryos. Percents are based on the proportion of embryos with the molecular phenotype indicated across triplicate experiments performed on three separate days.
Fig.7. In addition to the premature induction of cardiac field markers resulting from aberrant activation of GATA4 during gastrulation, the misregulation of Notch signaling results in altered levels of GATA4 transcription in gastrula stage embryos. Embryos were injected with one of the following constructs plus a pink lineage marker: GR-Su(H)VP16 (upregulate Notch signaling), GR-Su(H)DBM (downregulate Notch signaling), Esr9-GR (upregulate one Notch-responsive HES gene), or GATA4-GR (upregulate GATA4). Constructs were induced at stage 10 by the addition of dexamethasone to the culture medium, collected approximately 1 h later, and examined for GATA4, nkx2.5, or tbx5 expression via in situ hybridization. The predominant molecular phenotype (increased or decreased expression) is reported as the percentage of embryos with the observed phenotype calculated across three experiments. When Notch signaling was increased following injection and induction of either GR-Su(H)VP16 or Esr9-GR, GATA4 expression was reduced or ablated (71% of injected/induced embryos, n = 77; 74% of injected/induced embryos, n = 53, respectively). Unlike what is observed when Notch signaling is upregulated, when signaling through Notch was suppressed at these stages, GATA4 expression was expanded (76% of injected/induced embryos, n = 110). Interestingly when the temporally inducible GATA4-GR construct was induced at the onset of gastrulation, the expression of both nkx2.5 (83% of embryos, n = 42) and tbx5 (82% of embryos, n = 61) was activated prematurely in late-gastrula embryos. This phenotype is similar to that following suppression of Notch signaling (Fig. 6). For each construct used, injected embryos not exposed to hormone were also examined in order to determine the level of leakiness of the inducible constructs for these experiments. (B–D) Representative images of an uninjected-control embryo or embryos injected with GR-Su(H)VP16 or GR-Su(H)DBM demonstrating the observed GATA4 expression phenotypes. While GATA4 is detected in cells neighboring the dorsal lip of the blastopore (arrows in B), when Notch signaling is upregulated in cells in this region, GATA4 expression is reduced (arrowhead in C). Conversely, when Notch signaling is downregulated GATA4 is robustly expressed by migrating precardiac cells in addition to expression in cells lining the dorsal lip of the blastopore (arrowheads and arrows, respectively, in D). Embryos in panels B–C are oriented ventrally. The embryo in panel D is oriented laterally, with the blastopore facing down.