Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm

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.     

The mouse rib-vertebrae mutation disrupts anterior-posterior somite patterning and genetically interacts with a Delta1 null allele

Rib-vertebrae (rv) is an autosomal recessive mutation in mouse that affects the morphogenesis of the vertebral column. Axial skeleton defects vary along the anterior-posterior body axis, and include split vertebrae and neural arches, and fusions of adjacent segments. Here, we show that defective somite patterning underlies the vertebral malformations and altered Notch signaling may contribute to the phenotype. Somites in affected regions are irregular in size and shape, epithelial morphology is disrupted, and anterior-posterior somite patterning is abnormal, reminiscent of somite defects obtained in loss-of-function alleles of Notch signaling pathway components. Expression of Dll1, Dll3, Lfng and Notch1 is altered in rv mutant embryos, and rv and Dll1(lacZ), a null allele of the Notch ligand Delta1, genetically interact. Mice double heterozygous for rv and Dll1(lacZ), show vertebral defects, and one copy of Dll1(lacZ) on the homozygous rv background enhances the mutant phenotype and is lethal in the majority of cases. However, fine genetic mapping places rv into an interval on chromosome seven that does not contain a gene encoding a known component of the Notch signaling pathway.     

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.     

Expression of Notch receptors, ligands, and target genes during development of the mouse mammary gland

Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.     

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.     

An expanded domain of fgf3 expression in the hindbrain of zebrafish valentino mutants results in mis-patterning of the otic vesicle

The valentino (val) mutation in zebrafish perturbs hindbrain patterning and, as a secondary consequence, also alters development of the inner ear. We have examined the relationship between these defects and expression of fgf3 and fgf8 in the hindbrain. The otic vesicle in val/val mutants is smaller than normal, yet produces nearly twice the normal number of hair cells, and some hair cells are produced ectopically between the anterior and posterior maculae. Anterior markers pax5 and nkx5.1 are expressed in expanded domains that include the entire otic epithelium juxtaposed to the hindbrain, and the posterior marker zp23 is not expressed. In the mutant hindbrain, expression of fgf8 is normal, whereas the domain of fgf3 expression expands to include rhombomere 4 through rhombomere X (an aberrant segment that forms in lieu of rhombomeres 5 and 6). Depletion of fgf3 by injection of antisense morpholino (fgf3-MO) suppresses the ear patterning defects in val/val embryos: Excess and ectopic hair cells are eliminated, expression of anterior otic markers is reduced or ablated, and zp23 is expressed throughout the medial wall of the otic vesicle. By contrast, disruption of fgf8 does not suppress the val/val phenotype but instead interacts additively, indicating that these genes affect distinct developmental pathways. Thus, the inner ear defects observed in val/val mutants appear to result from ectopic expression of fgf3 in the hindbrain. These data also indicate that val normally represses fgf3 expression in r5 and r6, an interpretation further supported by the effects of misexpressing val in wild-type embryos. This is in sharp contrast to the mouse, in which fgf3 is normally expressed in r5 and r6 because of positive regulation by kreisler, the mouse ortholog of val. Implications for co-evolution of the hindbrain and inner ear are discussed.     

Expression of Notch pathway genes in the embryonic mouse metanephros suggests a role in proximal tubule development

The interaction of neighboring cells via Notch signalling leads to cell fate determination, differentiation and patterning of highly organized tissues. Mice with targeted disruption of genes from the Notch signal transduction pathway display defects in the developing somites, neurogenic structures, blood vessels, heart and other organs. Recent studies have added requirements for Notch signalling during kidney, pancreas and thymus morphogenesis. Here, we describe the expression of all four receptors (Notch1-4), the five transmembrane ligands (Dll1, 3, 4, Jag1 and Jag2), intracellular effectors (the Hey genes) and extracellular modulators (Lfng, Mfng, Rfng) in the developing mouse metanephros. Our results point to a Lfng-dependent role for Notch signalling in the development of nephron segments, especially the proximal tubules.     

New mutant mouse with skeletal deformities caused by mutation in delta like 3 (Dll3) gene.

We have established a new mouse strain with vertebral deformities caused by an autosomal single recessive mutation (oma). The mutant mice showed short trunk and short and kinky tail. The skeletal preparations of newborn and prenatal mice showed disorganized vertebrae and numerous vertebral and rib fusions which are thought to be caused by patterning defects at the stage of somitegenesis. Linkage analysis localized the oma locus on the proximal region of mouse chromosome 7 close to Dll3 gene. Dll3 is the gene involved in the Notch signaling pathway and null-mutation of the gene has been reported to cause vertebral deformities. The phenotypic similarity between oma and Dll3 null-mutant mice suggests that the causative gene for the oma mutant is the Dll3 gene. We, therefore, investigated the nucleotide sequence of the Dll3 gene of the oma mouse and found a single nucleotide substitution of G to T which causes missense mutation of glycine to cysteine at codon 409. Since the amino acid substitution is a nonconservative amino acid substitution at the conserved portion of the Dll3 protein, and the substitution is specific to the mutant mice, we concluded that the nucleotide substitution of the Dll3 gene is responsible for the skeletal deformities of the oma mouse.     

Dll3 pudgy mutation differentially disrupts dynamic expression of somite genes

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD.     

Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the Notch ligand Delta1

The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells. Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell. Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1). We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo. We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains. In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds. Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos.     

Periodic Delta-like 4 expression in developing retinal arteries

During vascular development, Notch signalling plays important roles in cell-cell communication and cell fate decisions. We studied expression of Notch 1-4 and its ligand Delta-like 4 (Dll4) in the developing retinal vasculature. Dll4 mRNA is strongly expressed in endothelial cells at the very tips of growing vessels ('tip cells') and also in arteries, where it is expressed in a segmented 'tiger's tail' pattern. This implies that developing retinal arteries contain different types of endothelial cells, Dll4-positive and Dll4-negative. The Dll4-positive stripes do not correspond to any obvious morphological property of the vascular network but correlate to some extent with the distribution of platelet derived growth factor B (PDGF-B) mRNA. However, PDGF-B expression is neither as artery-specific nor as clearly segmented as Dll4. Possible target cells for Dll4 signalling are retinal astrocytes (Notch1 positive), arterial pericytes (Notch3 positive) or arterial endothelial cells themselves (Notch4 positive). However, there is no clear reciprocity of Notch and Dll4 expression that allows identification of the interacting cells. Nevertheless, Dll4 stripes are a novel property of immature arteries, the origin and function of which remain to be explained.     

Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. We have investigated a potential role of Notch signaling and find that in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center.     

Fgfr2 and Fgfr3 are not required for patterning and maintenance of the midbrain and anterior hindbrain

The mid-/hindbrain organizer (MHO) is characterized by the expression of a network of genes, which controls the patterning and development of the prospective midbrain and anterior hindbrain. One key molecule acting at the MHO is the fibroblast growth factor (Fgf) 8. Ectopic expression of Fgf8 induces genes that are normally expressed at the mid-/hindbrain boundary followed by the induction of midbrain and anterior hindbrain structures. Inactivation of the Fgf receptor (Fgfr) 1 gene, which was thought to be the primary transducer of the Fgf8 signal at the MHO, in the mid-/hindbrain region, leads to a deletion of dorsal structures of the mid-/hindbrain region, whereas ventral tissues are less severely affected. This suggests that other Fgfrs might be responsible for ventral mid-/hindbrain region development. Here we report the analysis of Fgfr2 conditional knockout mice, lacking the Fgfr2 in the mid-/hindbrain region and of Fgfr3 knockout mice with respect to the mid-/hindbrain region. In both homozygous mouse mutants, patterning of the mid-/hindbrain region is not altered, neuronal populations develop normal and are maintained into adulthood. This analysis shows that the Fgfr2 and the Fgfr3 on their own are dispensable for the development of the mid-/hindbrain region. We suggest functional redundancy of Fgf receptors in the mid-/hindbrain region.     

O-Fucosylation of DLL3 Is Required for Its Function during Somitogenesis

Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis.