The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had ...identified two genes that cause a subset of autosomal recessive forms of this disease:
DLL3 (SCD1) and
MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene,
LUNATIC FRINGE (
LFNG), in a family with autosomal recessive SCD.
LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human
LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.
Spondylocostal dysostosis (SCD) is an inherited disorder that is characterized by the presence of extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic ...development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Previously, three genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681) and LFNG (SCDO3: MIM609813). These genes are all important components of the Notch signaling pathway, which has multiple roles in development and disease. Here we have used autozygosity mapping to identify a mutation in a fourth Notch pathway gene, HAIRY-AND-ENHANCER-OF-SPLIT-7 (HES7), in an autosomal recessive SCD family. HES7 encodes a bHLH-Orange domain transcriptional repressor protein that is both a direct target of the Notch signaling pathway, and part of a negative feedback mechanism required to attenuate Notch signaling. A missense mutation was identified in the DNA-binding domain of the HES7 protein. Functional analysis revealed that the mutant HES7 was not able to repress gene expression by DNA binding or protein heterodimerization. This is the first report of mutation in the human HES7 gene, and provides further evidence for the importance of the Notch signaling pathway in the correct patterning of the axial skeleton.
The Notch signalling pathway is widely utilised during embryogenesis in situations where cell–cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis ...into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction.
•Ligand binding induces Notch1 internalisation.•Ligand-dependent internalisation of Notch1 occurs via clathrin-mediated endocytosis.•Ligand is transendocytosed into Notch1-expressing cells.•Endocytosed Notch1 vesicles are SARA-positive.•Endocytosis is required in the Notch-expressing cell for signal transduction.
Notch4 is a divergent member of the Notch family of receptors that is primarily expressed in the vasculature. Its expression implies an important role for Notch4 in the vasculature; however, mice ...homozygous for the Notch4d1 knockout allele are viable. Since little is known about the role of Notch4 in the vasculature and how it functions, we further investigated Notch4 in mice and in cultured cells. We found that the Notch4d1 allele is not null as it expresses a truncated transcript encoding most of the NOTCH4 extracellular domain. In cultured cells, NOTCH4 did not signal in response to ligand. Moreover, NOTCH4 inhibited signalling from the NOTCH1 receptor. This is the first report of cis-inhibition of signalling by another Notch receptor. The NOTCH4 extracellular domain also inhibits NOTCH1 signalling when expressed in cis, raising the possibility that reported Notch4 phenotypes may not be due to loss of NOTCH4 function. To better address the role of NOTCH4 in vivo, we generated a Notch4 null mouse in which the entire coding region was deleted. Notch4 null mice exhibited slightly delayed vessel growth in the retina, consistent with our novel finding that NOTCH4 protein is expressed in the newly formed vasculature. These findings indicate a role of NOTCH4 in fine-tuning the forming vascular plexus.
•The Notch4d1 allele encodes for most of the NOTCH4 extracellular domain.•NOTCH4 lacks the capacity to signal in cultured cells.•NOTCH4 inhibits signalling through the NOTCH1 receptor.•Notch4−/− mice exhibit altered retinal angiogenesis.
Mouse delta-like 3 (Dll3), a novel vertebrate homologue of the Drosophila gene Delta was isolated by a subtracted library screen. In Drosphila, the Delta/Notch signalling pathway functions in many ...situations in both embryonic and adult life where cell fate specification occurs. In addition, a patterning role has been described in the establishment of the dorsoventral compartment boundary in the wing imaginal disc. Dll3 is the most divergent Delta homologue identified to date. We confirm that Dll3 can inhibit primary neurogenesis when ectopically expressed in Xenopus, suggesting that it can activate the Notch receptor and therefore is a functional Delta homologue. An extensive expression study during gastrulation and early organogenesis in the mouse reveals a diverse and dynamic pattern of expression. The three major sites of expression implicate Dll3 in somitogenesis and neurogenesis and in the production of tissue from the primitive streak and tailbud. A careful comparison of Dll3 and Dll1 expression by double RNA in situ hybridisation demonstrates that these genes have distinct patterns of expression, but implies that together they operate in many of the same processes. We postulate that during somitogenesis Dll3 and Dll1 coordinate in establishing the intersomitic boundaries. We confirm that, during neurogenesis in the spinal cord, Dll1 and Dll3 are expressed by postmitotic cells and suggest that expression is sequential such that cells express Dll1 first followed by Dll3. We hypothesise that Dll1 is involved in the release of cells from the precursor population and that Dll3 is required later to divert neurons along a specific differentiation pathway.
Normal development of the pituitary gland requires coordination between the maintenance of a progenitor cell pool and the selection of progenitor cells for differentiation. As Notch signaling ...controls progenitor cell differentiation in many embryonic tissues, we investigated the involvement of this important developmental pathway in the embryonic pituitary. We report that expression of Notch signaling genes is spatially and temporally regulated in pituitary embryogenesis and implicate
Notch2 in the differentiation of several cell lineages.
Notch2,
Notch3, and
Dll1 are initially expressed by most cells within the pituitary primordium and become restricted to a subset of the progenitor cell pool as differentiated pituitary cells begin to appear. Mutations in the transcription factor
Prop1 interfere with pituitary growth and cell specification, although the mechanism is unknown.
Notch2 expression is nearly absent in the developing pituitaries of
Prop1 mutant mice, but unaltered in some other panhypopituitary mutants, revealing that
Prop1 is directly or indirectly required for normal
Notch2 expression. Transgenic overexpression of
Prop1 is not sufficient for enhancement of endogenous
Notch2 expression, indicating that there are multiple inputs into this pathway.
Dll3 is expressed only in the presumptive corticotrope and melanotrope cells. Analysis of
Dll3 null mutants indicates that
Dll3 is not required for specification of these two cell types, although there may be functional overlap with
Dll1. The spatial and temporal expression patterns of Notch signaling genes in the pituitary suggest overlapping roles in pituitary growth and cell specification.
A loss-of-function mutation in the mouse delta-like3 (Dll3) gene has been generated following gene targeting, and results in severe axial skeletal defects. These defects, which consist of highly ...disorganised vertebrae and costal defects, are similar to those associated with the Dll3-dependent pudgy mutant in mouse and with spondylocostal dysplasia (MIM 277300) in humans. This study demonstrates that Dll3(neo) and Dll3(pu) are functionally equivalent alleles with respect to the skeletal dysplasia, and we suggest that the three human DLL3 mutations associated with spondylocostal dysplasia are also functionally equivalent to the Dll3(neo) null allele. Our phenotypic analysis of Dll3(neo)/Dll3(neo) mutants shows that the developmental origins of the skeletal defects lie in delayed and irregular somite formation, which results in the perturbation of anteroposterior somite polarity. As the expression of Lfng, Hes1, Hes5 and Hey1 is disrupted in the presomitic mesoderm, we suggest that the somitic aberrations are founded in the disruption of the segmentation clock that intrinsically oscillates within presomitic mesoderm.
Cited2 is widely expressed in the developing embryo and in extraembryonic tissues including the placenta. Gene expression can be induced by a number of factors; most notably by the hypoxia inducible ...transcription factor, HIF1, under low oxygen conditions.
Cited2 encodes for a transcriptional co-factor that in vitro can act as both a positive and negative regulator of transcription. This function is due to its interaction with CBP/p300 and appears to depend on whether Cited2 enables CBP/p300 to interact with the basic transcriptional machinery, or if its binding prevents such an interaction from occurring. Here, we report a novel function for
Cited2 in placenta formation, following gene deletion in mouse. In the absence of
Cited2 the placenta and embryo are significantly small from 12.5 and 14.5 dpc respectively, and death occurs in utero.
Cited2 null placentas have fewer differentiated trophoblast cell types; specifically there is a reduction in trophoblast giant cells, spongiotrophoblasts and glycogen cells. In addition, the fetal vasculature of the placenta is disorganised and there are fewer anastomosing capillaries. Given that
Cited2 is expressed in both trophoblasts and the fetal vasculature, the observed defects fit well with the sites of gene expression. We conclude that
Cited2 is required for normal placental development and vascularisation, and hence for embryo viability.
Establishment of the left-right axis is a fundamental process of vertebrate embryogenesis. Failure to develop left-right asymmetry leads to incorrect positioning and morphogenesis of numerous ...internal organs, and is proposed to underlie the etiology of several common cardiac malformations. The transcriptional modulator Cited2 is essential for embryonic development: Cited2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, exencephaly and adrenal agenesis. Cited2 is also required for normal establishment of the left-right axis; we demonstrate that abnormal heart looping and right atrial and pulmonary isomerism are consistent features of the left-right-patterning defect. We show by gene expression analysis that Cited2 acts upstream of Nodal, Lefty2 and Pitx2 in the lateral mesoderm, and of Lefty1 in the presumptive floor plate. Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.
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