Identification of genes involved in <i>Citif1</i> promoter activation and endoderm differentiation

In tadpole larva of the ascidian Ciona intestinalis (one of the most basal chordate) the endoderm is a simple tissue comprising about 500 cells. The commitment of endoderm occurs very early; in a 64-cells stage embryo five blastomeres pairs are restricted to endodermal fate. Although three genes, that exhibit an endoderm specific expression, Ci-alkaline phosphates, Cs-lhx3 and Cititf1 have been cloned, it remains to be elucidated how the first step of endoderm specification take place. As first strategy, I cloned a Ciona SoxE gene; this was found not to be endodermally expressed. In a second strategy, I focused on Cititf-1, a gene homologous to mammalian Nkx2.1 and the earliest zygotic endoderm specific marker isolated from Ciona thus far. Although its temporal and spatial expression seems to be finely regulated, the factors involved in its activation and likely involved in endoderm specification are unknown. The minimal region responsible for endodermal expression of Cititf1, has previously been located to a genomic fragment of about 200 base pairs upstream to putative TATA box sequence (Fanelli A. et al., 2003 Developmental Biology. 263, 12-23). Here I describe two imperfect repeats of eleven nucleotides, named R1 and R2, within this region and investigate their involvement in the transcriptional activation of Cititf1 promoter. Biochemical analyses showed that both repeats bind factors from ascidian embryos in a sequence-specific manner. The R2, however, is more important for endoderm specific expression and is able to drive the endodermal expression of a reporter gene. The R1 repeat cooperates with R2 to enhance this expression. A yeast one-hybrid screening was performed, leading to identification of a putative DNA-binding factor that could be involved in the regulation of Cititf1 in endoderm

TBX1 Represses <i>Vegfr2</i> Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells

<div><p>The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which <i>Tbx1</i> expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding <i>in vivo</i> during embryonic development. In addition, we found a <i>Vegfr2</i> domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of <i>Tbx1</i>. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of <i>Tbx1</i> expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after <i>Tbx1</i> pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells.</p></div

Expression profile of VEGFR2+ cells isolated at Day 4.75 of differentiation from mES-<i>Tbx1</i>Tet^Off cells and subjected to additional 48hrs in culture, with or without tetracycline.

<p>A: qRT-PCR assay of <i>Vegfr2</i> expression of cells bound to magnetic beads (VEGFR2+ cells) and unbound (UN). B: RT-PCR analysis of VEGFR2+ and unbound cells. C: qRT-PCR of <i>Tbx1</i> expression in VEGFR2+ cells. D,E: qRT-PCR evaluation of <i>Vegfr2</i> and <i>Smarcd3</i> expression in VEGFR2+ cells with and without Tet.</p

<i>Vegfr2</i> and <i>Tbx1</i> expression in E8.5 mouse embryos.

<p>A-D': In situ hybridization of <i>Tbx1</i> (A-D) or <i>Vegfr2</i> (A'-D') shown in a rostro-caudal series of transverse sections of wild type embryos. Arrows indicate <i>Vegfr2</i> expression in the pharyngeal mesenchyme. E-F: VEGFR2 immunohistochemistry in similar sections as in A and B. G-H: VEGFR2 immunohistochemistry in corresponding sections of a <i>Tbx1</i>^<i>-/-</i> embryo. Arrows indicate VEGFR2+ cells in the pharyngeal mesenchyme. OFT: cardiac outflow tract. IFT: cardiac inflow tract. Scale bars: 200 μm in A-D, A’-D’. 50 μm in E-H. s: number of somites.</p

Clonal analysis of VEGFR2+ cells.

<p>A: schematic representation of experimental strategy. B: Examples of RT-PCR analysis on VEGFR2+ cells in absence and in presence of <i>Tbx1</i> pulse. C: Color map panels of marker positivity of individual clones with or without <i>Tbx1</i> pulse. D: Graphic representation of clone multiplicity, based on the number of positive markers with and without tetracycline. Blue represents the percentage of clones that were positive for all three markers; Red represents the percentage of clones positive for two markers; Green represents the percentage of clones that are positive for one marker.</p

<i>Vegfr2</i> expression in wild type and <i>Tbx1</i>^-/- mouse embryos E9.5 and E9.0.

<p>A, A’: In situ hybridization of wild type and <i>Tbx1</i>^-/- embryos. B-C': Immunohistochemisty with an anti-VEGFR2 antibody on sagittal and transverse sections. The arrows indicate the expression domain in the pSHF. The arrowheads indicate the DMP region (n = number of embryos examined). Scale bars: 200 μm in A, A’, B, B’. 50 μm in C, C’. s: number of somites.</p

Differentiation assay of the mES-<i>Tbx1</i>Tet^OFF cell line.

<p>A: Scheme of experimental protocol. B: qRT-PCR assay of <i>Tbx1</i> expression in uninduced (+Tet) mES-<i>Tbx1</i>Tet ^OFF cells. A peak of expression is evident at day 6. C: Schematic representation of the experimental strategy used for pulse <i>Tbx1</i> expression. D: qRT-PCR analysis of <i>Tbx1</i> expression at day 6 after 24hrs without tetracycline. E: Immunofluorescence with an anti-P-H3 antibody on two colonies of mES-<i>Tbx1</i>Tet ^OFF cells with and without tetracycline at day 6. F: graphic representation of quantitative evaluation of mitotic activity using flow cytometry.</p

<i>Tbx1</i> expression pulse at day 5 produces persistent changes of expression of cardiac markers.

<p>A: qRT-PCR assay at Day 6, Day 8 and Day 11 of mES-<i>Tbx1</i>Tet ^OFF cells with (red) and without (blue) pulse at Day 5. The data are represented as mean of fold change relative to Day 5 value (not shown) of four different experiments. Error bars indicate SEM. (* = <i>p</i>-value<0.05; ** = <i>p</i>-value <0.005; *** = <i>p</i>-value <0.0005). B: Flow cytometric evaluation of the effects of <i>Tbx1</i> pulse on VEGFR2, NKX2.5 and GATA4 cell population at Day 8. Flow cytometry at Day 8 shows a decrease of the numbers of VEGFR2+ and GATA4+ cells (B, B”) and an increase of the number of NKX2.5+ cells (B’) after <i>Tbx1</i> pulse. C: The histograms show the mean percentage of positive cells in three different experiments. Error bar indicate SEM. D: Immunofluorescence detection of VEGFR2 with (Tet-) or without (Tet+) <i>Tbx1</i> pulse. FSC: Forward Scatter.</p

EZH2 is required for parathyroid and thymic development through differentiation of the third pharyngeal pouch endoderm

The Ezh2 gene encodes a histone methyltransferase of the Polycomb Repressive Complex 2 that methylates histone H3 lysine 27. In this work we asked whether EZH2 has a role in the development of the pharyngeal apparatus and whether it regulates the expression of the Tbx1 gene, which encodes a key transcription factor required in pharyngeal development. To these ends, we performed genetic in vivo experiments with mouse embryos and we used mouse embryonic stem cell (ESC)-based protocols to probe endoderm and cardiogenic mesoderm differentiation. Results showed that EZH2 occupies the Tbx1 gene locus in mouse embryos, and that suppression of EZH2 was associated with reduced expression of Tbx1 in differentiated mESCs. Conditional deletion of Ezh2 in the Tbx1 expression domain, which includes the pharyngeal endoderm, did not cause cardiac defects but revealed that the gene has an important role in the morphogenesis of the 3rd pharyngeal pouch (PP). We found that in conditionally deleted embryos the 3rd PP was hypoplastic, had reduced expression of Tbx1, lacked the expression of Gcm2, a gene that marks the parathyroid domain, but expressed FoxN1, a gene marking the thymic domain. Consistently, the parathyroids did not develop, and the thymus was hypoplastic. Thus, Ezh2 is required for parathyroid and thymic development, probably through a function in the pouch endoderm. This discovery also provides a novel interpretational key for the finding of Ezh2 activating mutations in hyperparathyroidism and parathyroid cancer

Vitamin B12 ameliorates the phenotype of a mouse model of DiGeorge syndrome

Pathological conditions caused by reduced dosage of a gene, such as gene haploinsufficiency, can potentially be reverted by enhancing the expression of the functional allele. In practice, low specificity of therapeutic agents, or their toxicity reduces their clinical applicability. Here, we have used a high throughput screening (HTS) approach to identify molecules capable of increasing the expression of the gene Tbx1, which is involved in one of the most common gene haploinsufficiency syndromes, the 22q11.2 deletion syndrome. Surprisingly, we found that one of the two compounds identified by the HTS is the vitamin B12. Validation in a mouse model demonstrated that vitamin B12 treatment enhances Tbx1 gene expression and partially rescues the haploinsufficiency phenotype. These results lay the basis for preclinical and clinical studies to establish the effectiveness of this drug in the human syndrome