DIO3 protects against thyrotoxicosis-derived cranio-encephalic and cardiac congenital abnormalities.

Maternal hyperthyroidism is associated with an increased incidence of congenital abnormalities at birth, but it is not clear which of these defects arise from a transient developmental excess of thyroid hormone and which depend on pregnancy stage, antithyroid drug choice, or unwanted subsequent fetal hypothyroidism. To address this issue, we studied a mouse model of comprehensive developmental thyrotoxicosis secondary to a lack of type 3 deiodinase (DIO3). Dio3-/- mice exhibited reduced neonatal viability on most genetic backgrounds and perinatal lethality on a C57BL/6 background. Dio3-/- mice exhibited severe growth retardation during the neonatal period and cartilage loss. Mice surviving after birth manifested brain and cranial dysmorphisms, severe hydrocephalus, choanal atresia, and cleft palate. These abnormalities were noticeable in C57BL/6J Dio3-/- mice at fetal stages, in addition to a thyrotoxic heart with septal defects and thin ventricular walls. Our findings stress the protecting role of DIO3 during development and support the hypothesis that human congenital abnormalities associated with hyperthyroidism during pregnancy are caused by transient thyrotoxicosis before clinical intervention. Our results also suggest thyroid hormone involvement in the etiology of idiopathic pathologies including cleft palate, choanal atresia, Chiari malformations, Kaschin-Beck disease, and Temple and other cranio-encephalic and heart syndromes.

Fine-tuning of Notch signaling sets the boundary of the organ of Corti and establishes sensory cell fates.

The signals that induce the organ of Corti and define its boundaries in the cochlea are poorly understood. We show that two Notch modifiers, Lfng and Mfng, are transiently expressed precisely at the neural boundary of the organ of Corti. Cre-Lox fate mapping shows this region gives rise to inner hair cells and their associated inner phalangeal cells. Mutation of Lfng and Mfng disrupts this boundary, producing unexpected duplications of inner hair cells and inner phalangeal cells. This phenotype is mimicked by other mouse mutants or pharmacological treatments that lower but not abolish Notch signaling. However, strong disruption of Notch signaling causes a very different result, generating many ectopic hair cells at the expense of inner phalangeal cells. Our results show that Notch signaling is finely calibrated in the cochlea to produce precisely tuned levels of signaling that first set the boundary of the organ of Corti and later regulate hair cell development.

Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure.

The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice.

DLL4/Notch1 and BMP9 Interdependent Signaling Induces Human Endothelial Cell Quiescence via P27KIP1 and Thrombospondin-1.

OBJECTIVE: Bone morphogenetic protein-9 (BMP9)/activin-like kinase-1 and delta-like 4 (DLL4)/Notch promote endothelial quiescence, and we aim to understand mechanistic interactions between the 2 pathways. We identify new targets that contribute to endothelial quiescence and test whether loss of Dll4(+/-) in adult vasculature alters BMP signaling. APPROACH AND RESULTS: Human endothelial cells respond synergistically to BMP9 and DLL4 stimulation, showing complete quiescence and induction of HEY1 and HEY2. Canonical BMP9 signaling via activin-like kinase-1-Smad1/5/9 was disrupted by inhibition of Notch signaling, even in the absence of exogenous DLL4. Similarly, DLL4 activity was suppressed when the basal activin-like kinase-1-Smad1/5/9 pathway was inhibited, showing that these pathways are interdependent. BMP9/DLL4 required induction of P27(KIP1) for quiescence, although multiple factors are involved. To understand these mechanisms, we used proteomics data to identify upregulation of thrombospondin-1, which contributes to the quiescence phenotype. To test whether Dll4 regulates BMP/Smad pathways and endothelial cell phenotype in vivo, we characterized the vasculature of Dll4(+/-) mice, analyzing endothelial cells in the lung, heart, and aorta. Together with changes in endothelial structure and vascular morphogenesis, we found that loss of Dll4 was associated with a significant upregulation of pSmad1/5/9 signaling in lung endothelial cells. Because steady-state endothelial cell proliferation rates were not different in the Dll4(+/-) mice, we propose that the upregulation of pSmad1/5/9 signaling compensates to maintain endothelial cell quiescence in these mice. CONCLUSIONS: DLL4/Notch and BMP9/activin-like kinase-1 signaling rely on each other's pathways for full activity. This represents an important mechanism of cross talk that enhances endothelial quiescence and sensitively coordinates cellular responsiveness to soluble and cell-tethered ligands.

Absence of a major role for the snai1 and snai3 genes in regulating skeletal muscle regeneration in mice.

The Snail gene family encodes DNA-binding zinc finger proteins that function as transcriptional repressors. While the Snai1 and Snai2 genes are required for normal development in mice, Snai3 mutant mice exhibit no obvious abnormalities. The Snai3 gene is expressed at high levels in skeletal muscle. However, we demonstrate by histological analysis that Snai3 null mutant mice exhibit normal skeletal muscle. During hindlimb muscle regeneration after cardiotoxin-mediated injury, the Snai3 null mice exhibited efficient regeneration. To determine whether the Snai3 gene functions redundantly with the Snai1 gene during skeletal muscle regeneration, we performed hindlimb muscle regeneration in mice with skeletal muscle-specific deletion of the Snai1 gene on a Snai3 null genetic background. These mice also exhibited efficient regeneration, demonstrating that there is no major role for the Snai1 and Snai3 genes in regulating skeletal muscle regeneration in mice.

The snail family gene snai3 is not essential for embryogenesis in mice.

The Snail gene family encodes zinc finger-containing transcriptional repressor proteins. Three members of the Snail gene family have been described in mammals, encoded by the Snai1, Snai2, and Snai3 genes. The function of the Snai1 and Snai2 genes have been studied extensively during both vertebrate embryogenesis and tumor progression and metastasis, and play critically important roles during these processes. However, little is known about the function of the Snai3 gene and protein. We describe here generation and analysis of Snai3 conditional and null mutant mice. We also generated an EYFP-tagged Snai3 null allele that accurately reflects endogenous Snai3 gene expression, with the highest levels of expression detected in thymus and skeletal muscle. Snai3 null mutant homozygous mice are viable and fertile, and exhibit no obvious phenotypic defects. These results demonstrate that Snai3 gene function is not essential for embryogenesis in mice.

The Notch-regulated ankyrin repeat protein is required for proper anterior-posterior somite patterning in mice.

The Notch-regulated ankyrin repeat protein (Nrarp) is a component of a negative feedback system that attenuates Notch pathway-mediated signaling. In vertebrates, the timing and spacing of formation of the mesodermal somites are controlled by a molecular oscillator termed the segmentation clock. Somites are also patterned along the rostral-caudal axis of the embryo. Here, we demonstrate that Nrarp-deficient embryos and mice exhibit genetic background-dependent defects of the axial skeleton. While progression of the segmentation clock occurred in Nrarp-deficient embryos, they exhibited altered rostrocaudal patterning of the somites. In Nrarp mutant embryos, the posterior somite compartment was expanded. These studies confirm an anticipated, but previously undocumented role for the Nrarp gene in vertebrate somite patterning and provide an example of the strong influence that genetic background plays on the phenotypes exhibited by mutant mice.

Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation.

The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1-deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis.

Segmentation defects of Notch pathway mutants and absence of a synergistic phenotype in lunatic fringe/radical fringe double mutant mice.

The Notch signaling pathway is important in regulating formation and anterior-posterior patterning of somites in vertebrate embryos. Here we show that distinct segmentation defects are displayed in embryos mutant for the Notch pathway genes Notch1, Lunatic fringe (Lfng), Delta-like 1 (Dll1), and Delta-like 3 (Dll3). Lfng-deficient mice and Dll3-deficient mice exhibit very similar defects, and marker analysis suggests that progression of the segmentation clock is disrupted in Dll3 mutants. We also show that Radical fringe (Rfng)-deficient mice exhibit no obvious phenotypic defects. To assess whether the absence of a phenotype in Rfng-deficient mice was the result of functional redundancy with the Lfng gene, we generated Lfng/Rfng double homozygous mutant mice. These mice exhibit the skeletal defects normally observed in Lfng-deficient mice, but we detected no obvious synergistic or additive effects in the double mutant animals.