Notch1 endocytosis is induced by ligand and is required for signal transduction.

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.

Notch4 reveals a novel mechanism regulating Notch 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 Notch4(d1) 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 Notch4(d1) 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.

Loss of Cited2 affects trophoblast formation and vascularization of the mouse placenta.

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.

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype.

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.

Breaking symmetry: a clinical overview of left-right patterning.

It is increasingly recognized that mutations in genes and pathways critical for left-right (L-R) patterning are involved in common isolated congenital malformations such as congenital heart disease, biliary tract anomalies, renal polycystic disease, and malrotation of the intestine, indicating that disorders of L-R development are far more common than a 1 in 10,000 incidence of heterotaxia might suggest. Understanding L-R patterning disorders requires knowledge of molecular biology, embryology, pediatrics, and internal medicine and is relevant to day-to-day clinical genetics practice. We have reviewed data from mammalian (human and mouse) L-R patterning disorders to provide a clinically oriented perspective that might afford the clinician or researcher additional insights into this diagnostically challenging area.

Developmental regulation of Notch signaling genes in the embryonic pituitary: Prop1 deficiency affects Notch2 expression.

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.

Kousseff syndrome: a causally heterogeneous disorder.

The existence of Kousseff syndrome as a distinct entity has been thrown into doubt by a recent study conducted on the family originally reported by Kousseff. In all cases where chromosome 22q11.2 FISH testing has been undertaken, including the original sibship, a chromosome 22q11.2-microdeletion has been identified. We report two cases of sacral myelomeningocele associated with a conotruncal cardiac anomaly or "Kousseff syndrome." The first case, a 4-year-old girl, had a sacral myelomeningocele, tetralogy of Fallot, microcephaly, hydrocephalus, hypoplasia of the corpus callosum and mild-moderate developmental delay. Chromosome 22q11.2 FISH was normal and the facial phenotype was not that of velocardiofacial syndrome. Sequencing of the entire coding region of CITED2 did not reveal a mutation. The second case, a male infant, was found to have a 22q11.2-microdeletion. These cases confirm Kousseff syndrome to be a causally heterogeneous disorder.

Dynamic expression patterns of the pudgy/spondylocostal dysostosis gene Dll3 in the developing nervous system.

Defects in the Notch pathway ligand Dll3 have been identified in the mouse pudgy (Dll3(pu)) and human spondylocostal dysostosis (SD, MIM 277300) mutations. Although these mutations are primarily associated with segmental defects in the axial skeleton and somitic patterning, they also exhibit cranial neurological defects. Therefore we have looked at the expression of Dll3 in the developing mouse nervous system. The expression of Notch ligands and receptors shares common features at 10.75 dpc in the rhombic lips and dorsal hindbrain. Temporal analysis of Dll3 expression from 9.0 to 11.0 dpc reveals that it is strongly expressed in laminar columns linked with regions of neuronal differentiation and hindbrain segmentation. Transverse sections show that Dll3 is expressed in territories where commissural neurons are formed. We have also looked at neuronal patterning in the mid-hindbrain region in Dll3(pu) mutants.

Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury.

We describe the identification, biochemical characterisation, and mutation of a novel mouse gene: Sp5. Sp5 encodes a protein having a C-terminal C(2)H(2) zinc finger domain closely related to that of the transcription factor Sp1. In vitro, DNA binding studies show that it binds to the GC box, a DNA motif present in the promoter of a very large number of genes, including Brachyury, and recognised by members of the Sp1 family. However, outside of its DNA binding domain, Sp5 has little homology with any other member of the Sp1 family. In contrast to the ubiquitous expression of Sp1, Sp5 exhibits a remarkably dynamic pattern of expression throughout early development. This is suggestive of a role in numerous tissue patterning events, including gastrulation and axial elongation; differentiation and patterning of the neural tube, pharyngeal region, and somites; and formation of skeletal muscle in the body and limbs. Mice homozygous for a targeted mutation in Sp5 show no overt phenotype. However, the enhancement of the T/+ phenotype in compound mutant mice (Sp5(lacZ)/Sp5(lacZ), T/+) indicates a genetic interaction between Sp5 and Brachyury. These observations are consistent with a role for Sp5 in the coordination of changes in transcription required to generate pattern in the developing embryo.

Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator.

Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor beta signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.

Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis.

Msg1 and Mrg1 are founding members of a gene family which exhibit distinct patterns of gene expression during mouse embryogenesis. Sequence analysis reveals that these genes are unlike any other gene identified to date, but they share two near-identical sequence domains. The Msg1 and Mrg1 expression profiles during early development are distinct from each other. Msg1 is predominantly expressed in nascent mesoderm, the heart tube, limb bud and sclerotome. Intriguingly, Msg1 expression is restricted, within these developing mesodermal sites, to posterior domains. Mrg1 is expressed prior to gastrulation in the anterior visceral endoderm. Expression is maintained in the endoderm once gastrulation has begun and commences in the rostralmost embryonic mesoderm which underlies the anterior visceral endoderm. Mrg1 expression persists in this rostral mesoderm as it is translocated caudalwards during the invagination of the foregut and the formation of the heart. Later Mrg1 expression predominates in the septum transversum caudal to the heart. This expression pattern suggests that the septum transversum originates from the rostralmost embryonic mesoderm which first expressed Mrg1 at the late primitive streak stage.

Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo.

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.

Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo.

A total of 5 conventional, directionally cloned plasmid cDNA libraries have been constructed from the entire embryonic region of the mid-gastrulation mouse embryo and from its four principal tissue constituents (ectoderm, mesoderm, endoderm and primitive streak). These libraries have been validated with respect to the number of independent clones, insert-size and appropriate representation of diagnostic marker genes. Subtractive hybridisation has been used to remove clones common to the Endoderm and Mesoderm cDNA libraries resulting in an Endoderm minus Mesoderm subtracted library. Probe prepared from this subtracted library has been hybridised to a grid containing approximately 18,500 Embryonic Region library clones. Three novel clones have been recovered as well as expected genes already known to be highly expressed in the primitive endoderm lineage at this stage of development. In situ hybridisation to early postimplantation embryos has revealed the expression patterns of these novel genes. One is highly expressed exclusively in visceral endoderm, one is expressed in ectodermal and endodermal tissues, and the third proves to be an early marker of prospective and differentiated surface ectoderm as well as being expressed in endoderm and its derivatives.

Multiple regions of the human cardiac actin gene are necessary for maturation-based expression in striated muscle.

In order to elucidate mechanisms involved in striated muscle contractile protein isoform expression, we have defined regulatory elements in the cardiac actin gene necessary for postnatal expression at the level of transcript accumulation in the heart and hindlimb muscles of transgenic mice. During this developmental period in the rodent, cardiac actin expression essentially remains constant in the heart, but declines significantly in skeletal muscle. We determined that a 13-kilobase human cardiac actin gene fragment contains sufficient information to direct this maturation-based developmental expression, as well as striated muscle-specific and high level expression. We localized an element responsible for maturation-based down-regulation in the 3' flank of the gene between approximately 950 and 2120 base pairs downstream of the polyadenylation site. Furthermore, we determined that -800 base pairs of 5'-flanking DNA, which contains multiple MyoD1 binding sites, as well as serum response element and AP1 binding sites, can account for striated muscle-specific expression, but not high level expression. Findings indicate that sequence(s) responsible for high level expression of the gene must be located within the body of the gene. We conclude that the human cardiac actin gene contains distinct sequences which confer developmental, tissue-specific, and high level expression.

Coordination of skeletal muscle gene expression occurs late in mammalian development.

The acquisition of specialized skeletal muscle fiber phenotypes during development is investigated by systematic measurement of the accumulation of 21 contractile protein mRNAs during hindlimb development in the rat and the human. During early myotube formation in both species there is no coordination of expression of either fast or slow contractile protein isoform genes, but rather some slow, some fast, and some cardiac isoforms are expressed. Some isoforms are not detected at all in early myotubes. From Embryonic Day 19 in the rat, and after 14 weeks in the human, a strong bias toward fast isoform expression is evident for all gene families examined. This results in the establishment of a coordinated fast isoform phenotype at birth in the rat, and by 24 weeks in the human fetus. Unexpectedly, during secondary myotube formation in the rat we observe sudden rises and falls in contractile protein gene output. We interpret these fluctuations in terms of periods of myoblast proliferation followed by synchronized fusion into myotubes. The data presented indicate that each contractile protein gene has its own determinants of mRNA accumulation and that the different myoblast populations which contribute to the developing limb are not intrinsically programmed to produce particular coordinated phenotypes with respect to the non-myosin heavy chain contractile proteins.