Raising the bar: The efficacy of bioorthogonal reactions for bioconjugation has been thoroughly evaluated in four different biological settings. Powered by development new biocompatible ligands, copper-catalyzed azide–alkyne cycloaddition (see picture) brought about unsurpassed efficiency, and thus it holds great promise as a highly potent adaptive tool broader spectrum applications. Detailed facts importance to specialist readers are published ”Supporting Information”. Such documents...

The Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand Cu(I), promotes reaction rapidly systems without apparent toxicity. This allows, first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing...

Summary Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development ovary some animals and testis others. The basis for this requirement not clear; most examined, both male female gonad express throughout life line. Here we characterized loss‐of‐function mutation zebrafish . We show that maternally provided stable through first ten days zebrafish, thus likely fulfills any early roles during germ‐line...

Mutations in Kinesin proteins (Kifs) are linked to various neurological diseases, but the specific and redundant functions of vertebrate Kifs incompletely understood. For example, Kif5A, not other Kinesin-1 heavy-chain family members, is implicated Charcot-Marie-Tooth disease (CMT) Hereditary Spastic Paraplegia (HSP), mechanism its involvement progressive axonal degeneration characteristic these diseases well We report that zebrafish kif5Aa mutants exhibit hyperexcitability, peripheral...

Although of fundamental importance in developmental biology, the genetic basis for symmetry breaking events that polarize vertebrate oocyte and egg are largely unknown. In vertebrates, first morphological asymmetry is Balbiani body, a highly conserved, transient structure found vertebrates invertebrates including Drosophila, Xenopus, human, mouse. We report identification zebrafish magellan (mgn) mutant, which exhibits novel enlarged body phenotype disruption polarity. To determine molecular...

In amphibian and teleost embryos, the dorsal determinants (DDs) are believed to be initially localized vegetal pole then transported prospective side of embryo along a microtubule array. The DDs known activate canonical Wnt pathway thereby promote expression genes that induce organizer. Here, by identifying locus maternal-effect ventralized mutant tokkaebi, we show Syntabulin, linker kinesin I motor protein, is essential for determination in zebrafish. We found syntabulin mRNA during...

In living color: Many mammalian glycans associated with signaling receptors contain terminal or penultimate N-acetyllactosamine. A highly specific method for labeling this disaccharide on cell-surface glycoproteins of live cultured cells and zebrafish embryos is reported. The two-step chemoenzymatic approach involves in situ fucosylation followed by a bioorthogonal click reaction (see scheme; α(1,3)FucT=α(1,3)-fucosyltransferase).

To monitor the kinetics of biological processes that take place within minute time scale, simple and fast analytical methods are required. In this article, we present our discovery an azide with internal Cu(I)-chelating motif enabled development fastest protocol for Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to date, its application toward following dynamic process glycan biosynthesis. We discovered electron-donating picolyl boosted efficiency ligand-accelerated CuAAC 20–38-fold in...

In vertebrates, the first asymmetries are established along animal-vegetal axis during oogenesis, but underlying molecular mechanisms poorly understood. Bucky ball (Buc) was identified in zebrafish as a novel vertebrate-specific regulator of oocyte polarity, acting through unknown interactions. Here we show that endogenous Buc protein localizes to Balbiani body, conserved, asymmetric structure oocytes requires for its formation. Asymmetric distribution precedes body formation, defining...

Summary: Convergence and extension are gastrulation movements that participate in the establishment of vertebrate body plan. Using new methods for quantifying convergence cell groups, we demonstrate wild-type embryos, dorsal lateral cells is initially slow, but speeds up between end gastrula period early segmentation. trilobite mutants normal during reduced by Morphometric studies revealed epiboly gastrulae become ovoid, whereas embryos remain rounder. By segmentation, exhibit shorter,...

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical electrical signals of nervous system. Although physiological properties cones rods well known, only a handful genes have been identified regulate specification photoreceptor subtypes. Taking advantage mosaic organization in zebrafish, we report isolation mutation resulting unique change cell fate. Mutation lots-of-rods (lor) locus results near one-for-one transformation...

Neue Maßstäbe: Die Wirksamkeit bioorthogonaler Reaktionen für die Biokonjugation wurde an vier biologischen Systemen getestet. Mit der Einführung neuer biokompatibler Liganden sind Kupfer-katalysierte Azid-Alkin-Cycloadditionen (siehe Schema) mit bisher unerreichter Biokonjugationseffizienz möglich, was Klick-Reaktion zu einer hoch wirksamen Methode ein breites Spektrum biologischer Anwendungen macht.

During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP system. Excess Gpr125 impaired C&E molecular polarities. Reduced function exacerbated facial branchiomotor neuron (FBMN) migration defects embryos with...

Live imaging of transcription and RNA dynamics has been successful in cultured cells tissues vertebrates but is challenging to accomplish vivo. The zebrafish offers important advantages study these processes--optical transparency during embryogenesis, genetic tractability rapid development. Therefore, an intact vertebrate organism, we have adapted the MS2 RNA-labeling system zebrafish. By using this binary coexpress a fluorescent bacteriophage coat protein (MCP) interest tagged with multiple...

Fertility and gamete reserves are maintained by asymmetric divisions of the germline stem cells to produce new or daughters that differentiate as gametes. Before entering meiosis, differentiating germ (GCs) sexual animals typically undergo cystogenesis. This evolutionarily conserved process involves synchronous incomplete mitotic a GC daughter (cystoblast) generate sister connected intercellular bridges facilitate exchange materials support rapid expansion progenitor population. Here, we...

Egg activation is an important cellular event required to prevent polyspermy and initiate development of the zygote. in all animals examined elicited by a rise free Ca2+ egg cytosol at fertilization. This crucial for subsequent steps, such as cortical granule exocytosis, which modifies vitelline membrane polyspermy. The cytosolic primarily initiated inositol 1,4,5-trisphosphate(IP3)-mediated release from endoplasmic reticulum. genes involved regulating IP3-mediated during remain largely...

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects the formation dorsoanterior structures. A positional cloning approach identified all mutations as stop codons affecting same gene, revealing that encodes Glutamate receptor interacting protein 2a (Grip2a), a containing multiple PDZ domains known to interact membrane-associated factors including components Wnt signaling pathway. We find grip2a mRNA...

In animals, specification of the primordial germ cells (PGCs), stem germline, is required to transmit genetic information from one generation next. Bucky ball (Buc) essential for plasm (GP) assembly in oocytes and its overexpression results excess PGCs zebrafish embryos. However, mechanistic basis response Buc overexpression, whether endogenous functions during embryogenesis are unknown. Here we show that Buc, like GP overexpressed Buc-GFP, accumulates at embryonic cleavage furrows....