We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

We have isolated a basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed zath3, from zebrafish. zath3 is expressed in neurons of the central nervous system and in subsets of cranial ganglia. Zebrafish mindbomb (mib) mutants have a higher density of zath3 expressing cells and narrowminded (nrd) mutants lack zath3 expression in a domain corresponding to primary sensory neurons showing that the expression of zath3 is regulated by both mib and nrd. Injection of synthetic zath3 RNA into zebrafish embryos expands the neural plate size, promotes ectopic expression of neuronal markers, and partially rescues the deficit of sensory neurons seen in nrd mutants. Interfering with zath3 function using antisense morpholino oligonucleotides (MO) has no significant effect on early neurogenesis. However, a double knock down of zath3 and neurogenin1 (ngn1), another atonal homologue, with morpholinos (MOs) leads to more severe defects in neurogenesis than are seen with ngn1 MO alone: a subtle reduction of motor and inter-neurons, and an almost complete loss all cranial ganglia. This study suggests that zath3 and ngn1 have partially overlapping roles in early neurogenesis.

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 trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans 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 trans fails but ability to inhibit in cis 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.

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.