IDENTIFICATION OF SIGNALING FACTORS INVOLVED IN THE REGULATION OF ALKALOID METABOLISM IN N. TABACUM

To identify the signaling mechanisms and components that are involved in regulation of a promoter for a gene involved in a secondary pathway I studied the nicotinic alkaloid biosynthetic pathway using various N. tabacum tissues. Nicotine and tropane alkaloids are widely known to be synthesized predominantly in the roots of species that produce pyrrolinium ring containing alkaloids. Putrescine Nmethyltransferase (PMT) catalyzes the first committed step in the biosynthesis of these alkaloid secondary products and earlier studies have indicated that PMT gene expression is restricted to root tissue in Solanaceae plants. To further elucidate the factors that govern the regulation of alkaloid synthesis, expression patterns dictated by the 5\u27-flanking region of one of the members of the PMT -gene family, NsPMT3, using the b-glucuronidase (GUS) reporter gene were examined. Various treatments were used to characterize the nature of signaling in various tissues of seedlings, whole plants and callus. High expression levels were detected in root tissue and no expression was detected in leaves, in agreement with previous studies. However, mechanically wounded leaves resulted in highly localized PMT expression. This wound-induced expression was transient, with maximum levels occurring immediately after wounding and diminishing after approximately 24 h. RT-PCR analysis of mRNA isolated from wild-type plants also indicated upregulation of PMT expression in leaves upon wounding as well as very low transcript levels in unwounded leaves. Low levels of PMT activity were detected in leaf tissue, and this activity did not increase significantly upon wounding. Transgenic callus material showed strong repression of PMT promoter activity in the presence of light and auxin, whereas dark conditions and the absence of auxin upregulated PMT promoter activity. Reactive oxygen species have been implicated in signaling. When treated with the scavengers of reactive oxygen species (ROS), dimethylthiourea (DMTU) or catalase, tobacco callus tissue, which displays highly repressed alkaloid synthesis under normal culture conditions in the light, exhibited significant induction of PMT promoter activity and alkaloid accumulation. It is thought that light repression signals through an ROS intermediate to affect changes in alkaloid pathway gene expression. Upregulation of PMT-promoter activity was observed upon treatment with JA (jasmonic acid) or darkness in roots of very young transgenic seedlings. Treatment with auxin, salicylic acid (SA) and H2O2, on the other hand, was found to highly repress PMT promoter activity. Action of other ROS such as nitric oxide and superoxide radicals on PMT expression is not clear but probably play less of a role, compared to H2O2. Consistent with this content ion, treatment with light or glucose oxidase (GOX) and glucose to generate H2O2, also repressed alkaloid accumulation, and treatment of seedlings to dark conditions, the ROS scavenger DMTU, or jasmonic acid resulted in alkaloid accumulation. Long distance signaling from leaves to roots is also suspected to involve ROS, as leaves treated with GOX and glucose exhibited repressed PMT promoter activity in roots. The responses of the PMT promoter to auxin, salicylic acid and H2O2 treatments were conserved as sho wn by similar responses of the N. tabacum PMT promoter when examined in transgenic Arabidopsis, thereby suggesting that these molecules signal through a conserved mechanism. Thus, ROS is strongly implicated in acting as an intermediate in these signaling processes with H2O2 proposed as a major signaling component

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans

Congenital heart disease-causing Gata4 mutation displays functional deficits in vivo.

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans

<i>Gata4 G295S</i> mutation has <i>in vivo</i> functional deficits in the early embryonic myocardium.

<p>(A) Embryonic lethality by E10.5 was found in compound heterozygote mice expressing only a <i>Gata4 G295S</i> mutant allele in early myocardium with the <i>Nkx2-5-Cre</i>, but normal Mendelian ratios were seen when the <i>Gata4 G295S</i> mutant allele was expressed in endocardium and late myocardium using <i>Tie2-Cre</i> and α-<i>MHC-Cre</i>, respectively. Images (B, D, F, H, J, L) and histologic sections (C, E, G, I, K, M) of E10.5 embryos generated with <i>Tie2-Cre</i>, which is specific for endocardium (B–E); α<i>-MHC-Cre</i>, which is specific for late embryonic myocardium (F–I); and <i>Nkx2.5-Cre</i>, which is specific for early embryonic myocardium (J–M), are shown. (L,M) Growth retardation and myocardial thinning were seen in <i>Gata4 G295S^ki/flox</i>; <i>Nkx2-5-Cre⁺</i> E10.5 embryos similar to the phenotype of the <i>Gata4 G295S^ki/ki</i> embryo. (H,I) The hearts of <i>Gata4 G295S^ki/flox</i>; α<i>-MHC-Cre⁺</i> appeared normal at E10.5. (D,E) While the <i>Gata4 G295S^ki/flox</i>; <i>Tie2-Cre⁺</i> did not show growth retardation or myocardial thinning, hypocellular endocardial cushions were noted (*). A, atria; V, ventricle; scale bars indicate 200 µm.</p

Targeting strategy for generation of <i>Gata4 G295S</i> knock-in mice.

<p>(A) Single nucleotide change resulting in the glycine to serine mutation was introduced into the mouse <i>Gata4</i> locus. Partial restriction map of the murine <i>Gata4</i> wildtype allele (top), the <i>Gata4</i> targeting vector (middle), and successfully targeted allele (bottom) are shown. Homologous recombination results in replacement of wildtype <i>Gata4</i> with genomic DNA harboring a substitution of glycine to serine at position 295 into the mouse <i>Gata4</i> locus, as well as the incorporation of neomycin cassette surrounded by loxP sites. <i>Gata4</i> coding exons are shown as empty boxes, whereas the exon used as a probe used for Southern blot analysis is highlighted by a black bar. NZf, amino- terminal zinc finger (exon 2); CZf, carboxy- terminal zinc finger (exon 3); E4, exon 4; E5, exon 5; E6, exon 6; B, BglI; S, SacI; E, EcoRV; and N, NotI. (B) Germline transmission of mutant allele was confirmed by Southern blotting after digestion of genomic DNA from <i>Gata4 G295S^ki/wt</i> and wiltype mice with BglI. A 3.8 kb wildtype band and a 12.5 kb mutant band using 3′ external probe are shown (black bar in A). (C) Direct sequencing confirmed the presence of mutated residue that altered glycine (GGC) to serine (AGC) in DNA from <i>Gata4 G295S^ki/ki</i> embryos. (D) Western blotting demonstrates that levels of Gata4 protein are equivalent in <i>Gata4 G295S^ki/ki</i> hearts (from three different embryos) as compared to wildtype E9.5 hearts. Equal protein loading is shown by Western blotting to GAPDH.</p

Atrial septal defects and semilunar valve stenosis in <i>Gata4 G295S^ki/wt</i> mice.

<p>(A) Table showing the frequency of cardiac abnormalities identified in <i>Gata4 G295S^ki/wt</i> mice (n = 9) and wildtype littermates (n = 12). Representative images of color (B, D, E, I, J) and pulsed wave Doppler (C, F, G, K, L) findings are shown. Small atrial communication demonstrated by both (B) color and (C) pulsed wave Doppler in <i>Gata4 G295S^ki/wt</i> mouse. Color Doppler recordings across normal aortic valve of a wildtype mouse (D) and stenotic aortic valve of <i>Gata4 G295S^ki/wt</i> mouse (E). Pulsed Doppler waveforms of flow across the aortic valve in wildtype (F) and <i>Gata4 G295S^ki/wt</i> (G) mice demonstrate increased aortic velocity in mutant mice. (H) Scatter plot showing aortic velocities in wildtype and <i>Gata4 G295S^ki/wt</i> mice. Four <i>Gata4 G295S^ki/wt</i> mice with aortic stenosis are indicated in red. Color Doppler recordings across pulmonary valve of a wildtype (I) and stenotic pulmonary valve in <i>Gata4 G295S^ki/wt</i> mice (J). Pulsed Doppler waveforms across pulmonary valve of wildtype (K) and <i>Gata4 G295S^ki/wt</i> mice (L) show increased velocity in mutant mice. (M) Scatter plot showing velocity across pulmonary valve in wildtype and <i>Gata4 G295S^ki/wt</i> mice. Two <i>Gata4 G295S^ki/wt</i> mice with pulmonary stenosis indicated in red.</p

Distribution of progeny obtained from intercrossing Gata4 G295S heterozygote mice.

<p>Distribution of progeny obtained from intercrossing Gata4 G295S heterozygote mice.</p

Decreased expression of Gata4 target genes in <i>Gata4 G295S^ki/ki</i> embryonic hearts.

<p>(A) Downregulation of ANF, α-MHC, cTnC, and Myl3 in homozygous mutant E9.5 hearts as compared to wildtype littermates as measured by qRT-PCR. *, p value<0.05. (B) Quantitative RT-PCR demonstrates no significant change in expression levels of Tbx5, Nkx2.5, Mef2C and β-MHC in E9.5 <i>Gata4 G295S^ki/ki</i> hearts when compared to wildtype littermates.</p

Proliferation deficits in <i>Gata4 G295S^ki/wt</i> embryonic cardiomyocytes.

<p>(A–I) Cells were isolated from wildtype (WT) and <i>Gata4 G295S^ki/wt</i> E11.5 and E13.5 hearts. FACS analyses for cardiac troponin T (cTnT)-positive cells was performed for (A, B) E11.5 atria, (C, D) E11.5 ventricles, (E, F) E13.5 atria, and (G,H) E13.5 ventricles. Proliferating cells are detected by staining with Ki67. Representative data are shown in each panel. (I) FACS analysis of unstained cells used as a control. Quantification of proliferative cardiomyocytes in <i>Gata4 G295S^ki/wt</i> mutant hearts as compared to wildtype littermate hearts at E11.5 (J) and E13.5 (K). Experiments were performed in triplicate using pooled hearts and all data are presented as means ± standard deviation; *<i>p</i> value<0.05. Coronal sections through the heart of wildtype (L, N) and <i>Gata4 G295S^ki/wt</i> (M, O) E12.5 hearts. High magnification images of the atria (L,M) and ventricle (N, O) are shown. Quantitative analysis demonstrates decreased wall thickness in the (P) atria and (Q) compact ventricular myocardium in <i>Gata4 G295S^ki/wt</i> as compared to wildtype littermates (n = 3 for each genotype). Arrowheads, representative site of measurement; *, <i>p</i> value<0.05. Scale bars indicate 200 µm.</p