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The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/β-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in β-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of β-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, β-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS., Author summary To understand the genetic relationship between Tbx1 and canonical Wnt/β-catenin, we performed gene expression profiling and genetic rescue experiments. We found that Tbx1 and β-catenin may provide a negative feedback loop to restrict premature differentiation in the anterior heart field. This is relevant to understanding the basis of variable expressivity of 22q11.2DS, caused by haploinsufficiency of TBX1.

Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish., Author Summary The somites—the embryonic segments of the vertebrate body—form one after another from tissue at the tail end of the embryo. A gene expression oscillator, the somite segmentation clock, operating in this tail region, marks out a periodic spatial pattern and so controls the segmentation process. Evidence from mutants shows that the Notch cell-cell signalling pathway has a critical role in the clock mechanism. However, when we switch on a blockade of Notch signalling, by immersing zebrafish embryos in the chemical inhibitor DAPT, the next ∼12 somites form normally, and only after that do disrupted somites appear. We show that this is because Notch signalling is needed only to maintain synchrony between the clocks of individual cells. The cells take about seven cycles to drift out of synchrony when Notch-mediated communication is blocked, and then a further five cycles to pass from the site where the tissue receives its “time-stamp” to the site where overt segmentation begins. By mathematical modelling, backed up with measurements on transgenic embryos, we show how Notch signalling may act at a molecular level to synchronise the intracellular oscillators of adjacent individual cells.