Tubulin glutamylation regulates axon guidance via the selective tuning of microtubule-severing enzymes.

The microtubule cytoskeleton is a major driving force of neuronal circuit development. Fine-tuned remodelling of this network by selective activation of microtubule-regulating proteins, including microtubule-severing enzymes, has emerged as a central process in neuronal wiring. Tubulin posttranslational modifications control both microtubule properties and the activities of their interacting proteins.

On the importance of evolutionary constraint for regulatory sequence identification.

Regulation of gene expression relies on the activity of specialized genomic elements, enhancers or silencers, distributed over sometimes large distance from their target gene promoters. A significant part of vertebrate genomes consists in such regulatory elements, but their identification and that of their target genes remains challenging, due to the lack of clear signature at the nucleotide level. For many years the main hallmark used for identifying functional elements has been their sequence conservation between genomes of distant species, indicative of purifying selection.

Creation of zebrafish knock-in reporter lines in the nefma gene by Cas9-mediated homologous recombination.

CRISPR/Cas9-based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)-induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock-ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9-targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence.

An translation-reporter system for the study of protein synthesis in zebrafish embryos.

Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique ( Lin et al.

BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.

Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in -encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling.

Initiation of cyp26a1 Expression in the Zebrafish Anterior Neural Plate by a Novel Cis-Acting Element.

Early patterning of the vertebrate neural plate involves a complex hierarchy of inductive interactions orchestrated by signalling molecules and their antagonists. The morphogen retinoic acid, together with the Cyp26 enzymes which degrade it, play a central role in this process. The cyp26a1 gene expressed in the anterior neural plate thus contributes to the fine modulation of the rostrocaudal retinoic acid gradient.

Zebrafish embryonic neurons transport messenger RNA to axons and growth cones in vivo.

Although mRNA was once thought to be excluded from the axonal compartment, the existence of protein synthesis in growing or regenerating axons in culture is now generally accepted. However, its extent and functional importance remain a subject of intense investigation. Furthermore, unambiguous evidence of mRNA axonal transport and local translation in vivo, in the context of a whole developing organism is still lacking.

DeltaC and DeltaD interact as Notch ligands in the zebrafish segmentation clock.

We describe the production and characterisation of two monoclonal antibodies, zdc2 and zdd2, directed against the zebrafish Notch ligands DeltaC and DeltaD, respectively. We use our antibodies to show that these Delta proteins can bind to one another homo- and heterophilically, and to study the localisation of DeltaC and DeltaD in the zebrafish nervous system and presomitic mesoderm (PSM). Our findings in the nervous system largely confirm expectations from previous studies, but in the PSM we see an unexpected pattern in which the localisation of DeltaD varies according to the level of expression of DeltaC: in the anterior PSM, where DeltaC is plentiful, the two proteins are colocalised in intracellular puncta, but in the posterior PSM, where DeltaC is at a lower level, DeltaD is seen mainly on the cell surface.

Setting the tempo in development: an investigation of the zebrafish somite clock mechanism.

The somites of the vertebrate embryo are clocked out sequentially from the presomitic mesoderm (PSM) at the tail end of the embryo. Formation of each somite corresponds to one cycle of oscillation of the somite segmentation clock--a system of genes whose expression switches on and off periodically in the cells of the PSM. We have previously proposed a simple mathematical model explaining how the oscillations, in zebrafish at least, may be generated by a delayed negative feedback loop in which the products of two Notch target genes, her1 and her7, directly inhibit their own transcription, as well as that of the gene for the Notch ligand DeltaC; Notch signalling via DeltaC keeps the oscillations of neighbouring cells in synchrony.

The vertebrate segmentation clock.

In vertebrate embryos, somite segmentation is controlled by a molecular clock, in the form of a transcriptional oscillator that operates in the presomitic mesoderm. Most of the genes implicated in the oscillator belong to the Notch pathway; a recently discovered exception is the Wnt pathway gene Axin2. Experiments have revealed several negative feedback loops that might generate oscillations, leading to at least four different theories.

c-jun regulation and function in the developing hindbrain.

Hindbrain development is a well-characterised segmentation process in vertebrates. The bZip transcription factor MafB/kreisler is specifically expressed in rhombomeres (r) 5 and 6 of the developing vertebrate hindbrain and is required for proper caudal hindbrain segmentation. Here, we provide evidence that the mouse protooncogene c-jun, which encodes a member of the bZip family, is coexpressed with MafB in prospective r5 and r6.

Novel activities of Mafb underlie its dual role in hindbrain segmentation and regional specification.

The bZip transcription factor Mafb is expressed in two segments of the developing vertebrate hindbrain: the rhombomeres 5 and 6. Loss of Mafb expression in the mouse mutant kreisler leads to elimination of r5 and to alterations of r6 regional identity. Here, we further investigated the role of Mafb in hindbrain patterning using gain-of-function experiments in the chick embryo.

Krox20 and kreisler co-operate in the transcriptional control of segmental expression of Hoxb3 in the developing hindbrain.

In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed at high relative levels in the rhombomeres (r) 5 and 6, and 5, respectively. The single enhancer elements responsible for these activities have been identified previously and shown to constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. Here, we have analysed the contribution of the transcription factor Krox20, present in r3 and r5.