Mir-125a-5p-mediated regulation of Lfng is essential for the avian segmentation clock.

Somites are embryonic precursors of the axial skeleton and skeletal muscles and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated at both the transcriptional and posttranscriptional levels for proper clock function. Here, we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3' UTR and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that microRNAs function in the posttranscriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of microRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight posttranscriptional regulation.

The Many Faces of MDM2 Binding Partners.

Mdm2 is an essential regulator of the p53 tumor suppressor. Mdm2 is modified at transcriptional, post-transcriptional, and post-translational levels to control p53 activity in normal versus stressed cells. Importantly, errors in these regulatory mechanisms can result in aberrant Mdm2 expression and failure to initiate programmed cell death in response to DNA damage. Such errors can have severe consequences as evidenced by tumor phenotypes resulting from amplification at the Mdm2 locus and changes in post-transcriptional and post-translational regulation of Mdm2. Although Mdm2 mediated inhibition of p53 is well characterized, Mdm2 interacts with many additional proteins and also targets many of these for proteosomal degradation. Mdm2 also has E3-ligase independent functions and p53-independent functions that have important implications for genome stability and cancer.

NOTCH1 missense alleles associated with left ventricular outflow tract defects exhibit impaired receptor processing and defective EMT.

Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1(G661S) and NOTCH1(A683T)) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1(A683T) exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1(G661S) exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.

NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling.

Congenital aortic valve stenosis (AVS), coarctation of the aorta (COA) and hypoplastic left heart syndrome (HLHS) are congenital cardiovascular malformations that all involve the left ventricular outflow tract (LVOT). They are presumably caused by a similar developmental mechanism involving the developing endothelium. The exact etiology for most LVOT malformations is unknown, but a strong genetic component has been established. We demonstrate here that mutations in the gene NOTCH1, coding for a receptor in a developmentally important signaling pathway, are found across the spectrum of LVOT defects. We identify two specific mutations that reduce ligand (JAGGED1) induced NOTCH1 signaling. One of these mutations perturbs the S1 cleavage of the receptor in the Golgi. These findings suggest that the levels of NOTCH1 signaling are tightly regulated during cardiovascular development, and that relatively minor alterations may promote LVOT defects. These results also establish for the first time that AVS, COA and HLHS can share a common pathogenetic mechanism at the molecular level, explaining observations of these defects co-occurring within families.