ABSTRACT
In canonical Delta-Notch signalling, expression of Delta activates Notch in neighbouring cells, leading to downregulation of Delta in these cells. This process of lateral inhibition results in selection of either Delta-signalling cells or Notch-signalling cells. Here we show that d-Asb11 is an important mediator of this lateral inhibition. In zebrafish embryos, morpholino oligonucleotide (MO)-mediated knockdown of d-Asb11 caused repression of specific Delta-Notch elements and their transcriptional targets, whereas these were induced when d-Asb11 was misexpressed. d-Asb11 also activated legitimate Notch reporters cell-non-autonomously in vitro and in vivo when co-expressed with a Notch reporter. However, it repressed Notch reporters when expressed in Delta-expressing cells. Consistent with these results, d-Asb11 was able to specifically ubiquitylate and degrade DeltaA both in vitro and in vivo. We conclude that d-Asb11 is a component in the regulation of Delta-Notch signalling, important in fine-tuning the lateral inhibition gradients between DeltaA and Notch through a cell non-autonomous mechanism.
Subject(s)
Membrane Proteins/metabolism , Receptors, Notch/metabolism , Signal Transduction , Suppressor of Cytokine Signaling Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Embryo, Nonmammalian/metabolism , Feedback, Physiological , Genes, Reporter , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Protein Binding , Transcriptional Activation/genetics , Zebrafish/embryologyABSTRACT
From a differential display designed to isolate genes that are down-regulated upon differentiation of the central nervous system in Danio rerio embryos, we isolated d-asb11 (ankyrin repeat and suppressor of cytokine signaling box-containing protein 11). Knockdown of the d-Asb11 protein altered the expression of neural precursor genes sox2 and sox3 and resulted in an initial relative increase in proneural cell numbers. This was reflected by neurogenin1 expansion followed by premature neuronal differentiation, as demonstrated by HuC labeling and resulting in reduced size of the definitive neuronal compartment. Forced misexpression of d-asb11 was capable of ectopically inducing sox2 while it diminished or entirely abolished neurogenesis. Overexpression of d-Asb11 in both a pluripotent and a neural-committed progenitor cell line resulted in the stimulus-induced inhibition of terminal neuronal differentiation and enhanced proliferation. We conclude that d-Asb11 is a novel regulator of the neuronal progenitor compartment size by maintaining the neural precursors in the proliferating undifferentiated state possibly through the control of SoxB1 transcription factors.
Subject(s)
Cell Differentiation/physiology , Central Nervous System/embryology , Central Nervous System/metabolism , Neurons/metabolism , Stem Cells/metabolism , Suppressor of Cytokine Signaling Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Amino Acid Sequence , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Lineage/physiology , Cell Proliferation , Central Nervous System/cytology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation/physiology , Gene Expression Regulation, Developmental/physiology , HMGB Proteins/metabolism , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Humans , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , PC12 Cells , Rats , SOXB1 Transcription Factors , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/isolation & purification , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/isolation & purificationABSTRACT
Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.
