The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis.

The Notch pathway is a highly conserved signaling system that controls a diversity of growth, differentiation, and patterning processes. In growing blood vessels, sprouting of endothelial tip cells is inhibited by Notch signaling, which is activated by binding of the Notch receptor to its ligand Delta-like 4 (Dll4). Here, we show that the Notch ligand Jagged1 is a potent proangiogenic regulator in mice that antagonizes Dll4-Notch signaling in cells expressing Fringe family glycosyltransferases. Upon glycosylation of Notch, Dll4-Notch signaling is enhanced, whereas Jagged1 has weak signaling capacity and competes with Dll4. Our findings establish that the equilibrium between two Notch ligands with distinct spatial expression patterns and opposing functional roles regulates angiogenesis, a mechanism that might also apply to other Notch-controlled biological processes.

The FOXJ1 target Cfap206 is required for sperm motility, mucociliary clearance of the airways and brain development.

Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.

CFAP43 modulates ciliary beating in mouse and Xenopus.

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.

CFAP157 is a murine downstream effector of FOXJ1 that is specifically required for flagellum morphogenesis and sperm motility.

Motile cilia move extracellular fluids or mediate cellular motility. Their function is essential for embryonic development, adult tissue homeostasis and reproduction throughout vertebrates. FOXJ1 is a key transcription factor for the formation of motile cilia but its downstream genetic programme is only partially understood. Here, we characterise a novel FOXJ1 target, Cfap157, that is specifically expressed in motile ciliated tissues in mouse and Xenopus in a FOXJ1-dependent manner. CFAP157 protein localises to basal bodies and interacts with tubulin and the centrosomal protein CEP350. Cfap157 knockout mice appear normal but homozygous males are infertile. Spermatozoa display impaired motility and a novel phenotype: Cfap157-deficient sperm exhibit axonemal loops, supernumerary axonemal profiles with ectopic accessory structures, excess cytoplasm and clustered mitochondria in the midpiece regions, and defective axonemes along the flagella. Our study thus demonstrates an essential sperm-specific function for CFAP157 and suggests that this novel FOXJ1 effector is part of a mechanism that acts during spermiogenesis to suppress the formation of supernumerary axonemes and ensures a correct ultrastructure.

Identification of FOXJ1 effectors during ciliogenesis in the foetal respiratory epithelium and embryonic left-right organiser of the mouse.

Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap - including 1700012B09Rik, 1700026L06Rik and Fam183b - we provide extended experimental evidence for a ciliary function.

1700012B09Rik, a FOXJ1 effector gene active in ciliated tissues of the mouse but not essential for motile ciliogenesis.

In humans and mice, motile cilia occur on the surface of the embryonic ventral node, on respiratory and ependymal epithelia and in reproductive organs where they ensure normal left-right asymmetry of the organism, mucociliary clearance of airways, homeostasis of the cerebrospinal fluid and fertility. The genetic programme for the formation of motile cilia, thus critical for normal development and health, is switched on by the key transcription factor FOXJ1. In previous microarray screens for murine FOXJ1 effectors, we identified candidates for novel factors involved in motile ciliogenesis, including both genes that are well conserved throughout metazoa and beyond, like FOXJ1 itself, and genes without overt homologues outside higher vertebrates. Here we examine one of the novel murine FOXJ1 effectors, the uncharacterised 1700012B09Rik whose homologues appear to be restricted to higher vertebrates. In mouse embryos and adults, 1700012B09Rik is predominantly expressed in motile ciliated tissues in a FOXJ1-dependent manner. 1700012B09RIK protein localises to basal bodies of cilia in cultured cells. Detailed analysis of 1700012B09RiklacZ knock-out mice reveals no impaired function of motile cilia or non-motile cilia. In conclusion, this novel FOXJ1 effector is associated mainly with motile cilia but - in contrast to other known FOXJ1 targets - its putative ciliary function is not essential for development or health in the mouse, consistent with a late emergence during evolution of motile ciliogenesis.

Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo.

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.

A novel Cre-inducible knock-in ARL13B-tRFP fusion cilium reporter.

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre-inducible cilium-specific reporter mouse line expressing an ARL13B-tRFP fusion protein driven by a CMV enhancer/chicken ß actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono- and multiciliated tissues and by time-lapse live-cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live-cell imaging. Thus, the ARL13B-tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue-specific manner to understand processes, such as ciliary protein trafficking or cilium-dependent signaling in vitro and in vivo.

Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis.

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

Differential regulation of node formation, nodal ciliogenesis and cilia positioning by Noto and Foxj1.

The mouse transcription factor Noto is expressed in the node and controls node morphogenesis, formation of nodal cilia and left-right asymmetry. Noto acts upstream of Foxj1, which regulates ciliogenesis in other mouse tissues. However, the significance of Foxj1 for the formation of cilia in the mouse node is unclear; in non-amniote species Foxj1 is required for ciliogenesis in the structures equivalent to the node. Here, we analyzed nodes, nodal cilia and nodal flow in mouse embryos in which we replaced the Noto-coding sequence with that of Foxj1, or in embryos that were deficient for Foxj1. We show that Foxj1 expressed from the Noto locus is functional and restores the formation of structurally normal motile cilia in the absence of Noto. However, Foxj1 is not sufficient for the correct positioning of cilia on the cell surface within the plane of the nodal epithelium, and cannot restore normal node morphology. We also show that Foxj1 is essential for ciliogenesis upstream of Rfx3 in the node. Thus, the function of Foxj1 in vertebrate organs of asymmetry is conserved, and Noto regulates node morphogenesis and the posterior localization of cilia on node cells independently of Foxj1.

Generation of an 870 kb deletion encompassing the Skt/Etl4 locus by combination of inter- and intra-chromosomal recombination.

BACKGROUND: Etl4(lacZ) (Enhancer trap locus 4) and Skt(Gt) (Sickle tail) are lacZ reporter gene integrations into the same locus on mouse chromosome 2 targeting a gene that is expressed in the notochord of early embryos and in multiple epithelia during later development. Both insertions caused recessive mutations that resulted exclusively in mild defects in the caudal vertebral column. Since notochord-derived signals are essential for formation of the vertebral column the phenotypes suggested that the lacZ insertions interfered with some notochord-dependent aspect of vertebral development. As both insertions occurred in introns it was unclear whether they represent hypomorphic alleles or abolish gene function. Here, we have generated a definitive null allele of the Skt/Etl4 gene and analysed homozygous mutants. RESULTS: We have introduced loxP sites into three positions of the gene based on additional upstream exons that we identified, and deleted approximately 870 kb of the locus by a combination of inter- and intra-chromosomal Cre-mediated recombinations in the female germ line of mice. This deletion removes about 90 % of the coding region and results in the loss of the SKT/ETL4 protein. Similar to the Etl4(lacZ) and Skt(Gt) alleles our deletion mutants are viable and fertile and show only mild defects in caudal vertebrae due to abnormal intervertebral disc development, although with higher penetrance. No other tissue with Skt/Etl4 expression that we analysed showed obvious defects. CONCLUSION: The complete loss of Skt/Etl4 function affects only development of caudal notochord derivatives and is compensated for in its other expression domains.

Live imaging and genetic analysis of mouse notochord formation reveals regional morphogenetic mechanisms.

The node and notochord have been extensively studied as signaling centers in the vertebrate embryo. The morphogenesis of these tissues, particularly in mouse, is not well understood. Using time-lapse live imaging and cell lineage tracking, we show the notochord has distinct morphogenetic origins along the anterior-posterior axis. The anterior head process notochord arises independently of the node by condensation of dispersed cells. The trunk notochord is derived from the node and forms by convergent extension. The tail notochord forms by node-derived progenitors that actively migrate toward the posterior. We also reveal distinct genetic regulation within these different regions. We show that Foxa2 compensates for and genetically interacts with Noto in the trunk notochord, and that Noto has an evolutionarily conserved role in regulating axial versus paraxial cell fate. Therefore, we propose three distinct regions within the mouse notochord, each with unique morphogenetic origins.

Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo.

The Notch ligands Dll1 and Dll3 are coexpressed in the presomitic mesoderm of mouse embryos. Despite their coexpression, mutations in Dll1 and Dll3 cause strikingly different defects. To determine if there is any functional equivalence, we replaced Dll1 with Dll3 in mice. Dll3 does not compensate for Dll1; DLL1 activates Notch in Drosophila wing discs, but DLL3 does not. We do not observe evidence for antagonism between DLL1 and DLL3, or repression of Notch activity in mice or Drosophila. In vitro analyses show that differences in various domains of DLL1 and DLL3 individually contribute to their biochemical nonequivalence. In contrast to endogenous DLL1 located on the surface of presomitic mesoderm cells, we find endogenous DLL3 predominantly in the Golgi apparatus. Our data demonstrate distinct in vivo functions for DLL1 and DLL3. They suggest that DLL3 does not antagonize DLL1 in the presomitic mesoderm and warrant further analyses of potential physiological functions of DLL3 in the Golgi network.

Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates.

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif.

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. Here, we demonstrate that the proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. We identify by in silico screening a number of additional candidate target genes, which are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. We thus propose that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis.

DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries.

Notch signaling has been shown to regulate various aspects of vascular development. However, a specific role of the ligand Delta-like 1 (DLL1) has not been shown thus far. Here, we demonstrate that during fetal development, DLL1 is an essential Notch ligand in the vascular endothelium of large arteries to activate Notch1 and maintain arterial identity. DLL1 was detected in fetal arterial endothelial cells beginning at embryonic day 13.5. While DLL4-mediated activation has been shown to suppress vascular endothelial growth factor (VEGF) pathway components in growing capillary beds, DLL1-Notch signaling was required for VEGF receptor expression in fetal arteries. In the absence of DLL1 function, VEGF receptor 2 (VEGFR2) and its coreceptor, neuropilin-1 (NRP1), were down-regulatedin mutant arteries, which was followed by up-regulation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a repressor of arterial differentiation and Nrp1 expression in veins. Consistent with a positive modulation of the VEGF pathway by DLL1, the Nrp1 promoter contains several recombinant signal-binding protein 1 for J kappa (RBPJkappa)-binding sites and was responsive to Notch activity in cell culture. Our results establish DLL1 as a critical endothelial Notch ligand required for maintaining arterial identity during mouse fetal development and suggest context-dependent interrelations of the VEGFA and Notch signaling pathways.

Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo.

OBJECTIVE: The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. RESULTS: We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

The highly conserved FOXJ1 target CFAP161 is dispensable for motile ciliary function in mouse and Xenopus.

Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity.

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.