Katrin Serth

Specification of vertebral identity is coupled to Notch signalling and the segmentation clock

To further analyse requirements for Notch signalling in patterning the paraxial mesoderm, we generated transgenic mice that express in the paraxial mesoderm a dominant-negative version of Delta1. Transgenic mice with reduced Notch activity in the presomitic mesoderm as indicated by loss of Hes5 expression were viable and displayed defects in somites and vertebrae consistent with known roles of Notch signalling in somite compartmentalisation. In addition, these mice showed with variable expressivity and penetrance alterations of vertebral identities resembling homeotic transformations, and subtle changes of Hox gene expression in day 12.5 embryos. Mice that carried only one functional copy of the endogenous Delta1 gene also showed changes of vertebral identities in the lower cervical region, suggesting a previously unnoticed haploinsufficiency for Delta1. Likewise, in mice carrying a null allele of the oscillating Lfng gene, or in transgenic mice expressing Lfng constitutively in the presomitic mesoderm, vertebral identities were changed and numbers of segments in the cervical and thoracic regions were reduced, suggesting anterior shifts of axial identity. Together, these results provide genetic evidence that precisely regulated levels of Notch activity as well as cyclic Lfng activity are critical for positional specification of the anteroposterior body axis in the paraxial mesoderm.

O-fucosylation of the notch ligand mDLL1 by POFUT1 is dispensable for ligand function.

Fucosylation of Epidermal Growth Factor-like (EGF) repeats by protein O-fucosyltransferase 1 (POFUT1 in vertebrates, OFUT1 in Drosophila) is pivotal for NOTCH function. In Drosophila OFUT1 also acts as chaperone for Notch independent from its enzymatic activity. NOTCH ligands are also substrates for POFUT1, but in Drosophila OFUT1 is not essential for ligand function. In vertebrates the significance of POFUT1 for ligand function and subcellular localization is unclear. Here, we analyze the importance of O-fucosylation and POFUT1 for the mouse NOTCH ligand Delta-like 1 (DLL1). We show by mass spectral glycoproteomic analyses that DLL1 is O-fucosylated at the consensus motif C2XXXX(S/T)C3 (where C2 and C3 are the second and third conserved cysteines within the EGF repeats) found in EGF repeats 3, 4, 7 and 8. A putative site with only three amino acids between the second cysteine and the hydroxy amino acid within EGF repeat 2 is not modified. DLL1 proteins with mutated O-fucosylation sites reach the cell surface and accumulate intracellularly. Likewise, in presomitic mesoderm cells of POFUT1 deficient embryos DLL1 is present on the cell surface, and in mouse embryonic fibroblasts lacking POFUT1 the same relative amount of overexpressed wild type DLL1 reaches the cell surface as in wild type embryonic fibroblasts. DLL1 expressed in POFUT1 mutant cells can activate NOTCH, indicating that POFUT1 is not required for DLL1 function as a Notch ligand.

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.

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

ABSTRACT Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.

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. Summary: Loss of Cfap157, a novel target of FOXJ1 in cells carrying motile cilia, causes the formation of supernumerary and abnormal axonemes in mouse sperm and leads to male infertility.

Tumor Specific Epigenetic Silencing of Corticotropin Releasing Hormone -Binding Protein in Renal Cell Carcinoma: Association of Hypermethylation and Metastasis.

The relevance of Corticotropin Releasing Hormone (CRH)-system in human malignancies is a question of growing interest. Here we investigated hypermethylation and epigenetic silencing of the CRH-Binding Protein (CRHBP) gene in clear cell renal cell cancer (ccRCC). Relative methylation of the CRHBP CpG island (CGI) was determined in 17 tumor cell lines as well as 86 ccRCC samples and 66 paired normal tissues using pyrosequencing and quantitative methylation specific PCR of bisulfite converted DNA. Results were statistically compared with relative mRNA expression levels of CRHBP and clinicopathological parameters of patients. Re-expression of CRHBP following 5-aza-2´-deoxycytidine treatment was investigated by quantitative mRNA expression analysis. Real-time impedance analysis was applied for analysis of invasiveness of renal tumor cells following si-RNA knockdown of CRHBP expression or ectopic expression of CRHBP. We found the CRHBP CGI to be frequently methylated in tumor cell lines of renal, prostatic, and bladder cancer. Comparison of methylation in normal and paired renal cancer tissue specimens revealed hypermethylation of the CRHBP CGI in tumors (p<1*10−12). DNA methylation and decreased mRNA expression were correlated (R = 0.83, p<1*10−12). Tumor cell lines showed 5-aza-2´-deoxycytidine dependent reduction of methylation and re-expression of CRHBP was associated with altered cellular invasiveness of renal cancer cells in real-time impedance invasion assays. Hypermethylation and inverse relationship with mRNA expression were validated in silico using the TCGA network data. We describe for the first time tumor specific epigenetic silencing of CRHBP and statistical association with aggressive tumors thus suggesting the CRH system to contribute to the development of kidney cancer.

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

Geffers et al. 2007. J.Cell Biol. doi:10.1083/jcb.200702009 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200702009%26rft_id%253Dinfo%253Apmid%252F17664336%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%