Endocytosis of DeltaD deletion mutants.

<p>(A,E,I,M,Q,U) Distribution of zdd2 (green) in COS7 cells transfected with DeltaD (A) or DeltaD ∆A, ∆B, ∆C, ∆D or ∆A-D deletion mutants (E, I, M, Q, U). Surface DeltaD was first labelled by incubation with zdd2 at 4°C for 30’ then, following washout of unbound zdd2, internalization was allowed for 30’ at 37°C. Nuclei were labelled with DAPI (blue). (B-D, F-H, J-L, N-P, R-T, V-X) Distribution of zdd2 (green) in COS7 cells co-transfected with DeltaD constructs and Mib1 (red) following internalization as described above. Each set of 3 panels, respectively, shows distribution of the DeltaD construct (green), Myc-Mib1 (red)/nuclei (blue), and the merged image. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127864#sec002" target="_blank">materials and methods</a> for details.</p

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD

<div><p>Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in <i>trans</i>) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in <i>cis</i>). As a result, forms of DeltaD that have the MID can activate Notch in <i>trans</i> and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in <i>trans</i> fails but ability to inhibit in <i>cis</i> is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.</p></div

Identification of the <i>Mib1</i>-interacting domain (MID) in the Notch ligand DeltaD.

<p>(A) DeltaD deletion constructs and point mutations. Asterisks represent relative positions of NN and KNxNKK motifs. (B) Mib1 does not interact effectively with DeltaD ∆B-D. Myc-Mib1 was co-immunoprecipitated with full-length DeltaD and truncation mutants (∆D, ∆C-D, ∆B-D) using zdd2 antibody (Ab) and detected with anti-Myc Ab. (C) ∆B-D is not effectively ubiquitylated by Mib1. Full-length and DeltaD truncation mutants, co-transfected with HA-ubiquitin (HA-Ub), with and without Myc-Mib1, were immunoprecipitated with zdd2 Ab and immunoblotted with anti-HA Ab to detect ubiquitylated DeltaD. (D) Delta ∆A (∆A) and Delta ∆B (∆B) interact poorly with Mib1. HA-tagged DeltaD and deletion constructs co-transfected with and without Myc-Mib1 are immunoprecipitated with anti-Myc Ab and detected with anti-HA Ab. Relative density of IP anti-Myc band normalized to lysate anti-HA band. (E) ∆B is not effectively ubiquitylated by Mib1. DeltaD-HA and deletion constructs are immunoprecipitated with anti-HA Ab to detect total ubiquitylated DeltaD with and without Myc-Mib1.</p

Identification of critical residues in the Mib1-Interacting Domain (MID).

<p>(A) Conserved amino acids (pink shading) in the putative <i>Mib1</i>-Interacting Domain in Delta ligands. (B) KK and NN/KK mutants do not significantly interact with mind bomb. DeltaD-HA, ∆B and point mutation (NN, KK and NN/KK) constructs are co-immunoprecipitated with Myc-Mib1 using anti-Myc Ab and detected with anti-HA Ab. (C) KK and NN/KK mutants are not significantly ubiquitylated by Mib1. DeltaD-HA, ∆B and point mutation constructs co-transfected with Flag-Ubiquitin (Flag-Ub) with and without Myc-Mib1 are immunoprecipitated with anti-HA Ab and detected with Anti-Flag Ab to assay ubiquitylation of full length and mutant forms of DeltaD.</p

Ectopic expression of deltaD deletion and point mutant recapitulates neurogenic phenotype of DeltaD (∆A-D).

<p>(A) The prospective distribution of neurons revealed by the distribution of <i>huC</i> as revealed by <i>in situ</i> hybridization probe (purple) in control embryos injected with only <i>ß-galactosidase</i> mRNA. (B-F) <i>huC</i> in embryos co-injected with <i>ß-galactosidase</i> and <i>deltaD</i> (B), <i>deltaD-∆C</i> (C), <i>deltaD ∆B</i> (D), <i>deltaD NN/KK</i> (E) or <i>DeltaD ∆A-D</i> (F) mRNA. Distribution of ectopic mRNA injected in one cell at the two-cell stage revealed by X-Gal distribution (blue). Dorsal view, rostral to the left. Embryos are at approximately the 3 somite stage. (G) Quantification of the effect of ectopic expression of mRNA encoding various forms of DeltaD on the distribution of early neurons. Red indicates fraction with a neurogenic phenotype (increased density of neurons), Green—fraction with no obvious effect on neuron density, Blue- fraction with suppression of neurogenesis (reduced neuron density). P-values for pairwise comparison based on Fisher’s Exact test of independence. P >. 05 does not meet the criteria for the Null hypothesis that pairs contain an equivalent distribution of phenotype classes.</p

Polarization and migration in the zebrafish posterior lateral line system

<div><p>Collective cell migration plays an important role in development. Here, we study the posterior lateral line primordium (PLLP) a group of about 100 cells, destined to form sensory structures, that migrates from head to tail in the zebrafish embryo. We model mutually inhibitory FGF-Wnt signalling network in the PLLP and link tissue subdivision (Wnt receptor and FGF receptor activity domains) to receptor-ligand parameters. We then use a 3D cell-based simulation with realistic cell-cell adhesion, interaction forces, and chemotaxis. Our model is able to reproduce experimentally observed motility with leading cells migrating up a gradient of CXCL12a, and trailing (FGF receptor active) cells moving actively by chemotaxis towards FGF ligand secreted by the leading cells. The 3D simulation framework, combined with experiments, allows an investigation of the role of cell division, chemotaxis, adhesion, and other parameters on the shape and speed of the PLLP. The 3D model demonstrates reasonable behaviour of control as well as mutant phenotypes.</p></div