Methods for Visualization of RNA and Cytoskeletal Elements in the Early Zebrafish Embryo

Hansen, Christina L; Pelegri, Francisco

doi:10.1007/978-1-0716-0970-5_18

Cited by 4 publications

(1 citation statement)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Embryos were allowed to hybridize overnight at 69 °C with RNA probes labeled with either fluorescein‐ (FLU‐), digoxigenin‐ (DIG‐), or dinitrophenol‐ (DNP‐) conjugated UTPs. FLU, DIG, and DNP haptens were detected and fluorescent signal was developed using the TSA Plus Fluorescein/Cy3/Cy5 system (PerkinElmer, Waltham, MA) following a modified “Triple fluorescent in situ” protocol (Hansen & Pelegri, 2021a). Following completion of the FISH procedure, embryos were stained to detect DNA with 1:200 dilution of 100 μg/ml DAPI in PBS for 10 min followed by PBS washes.…”

Section: Methodsmentioning

confidence: 99%

Conserved germ plasm characteristics across theDanioandDevariolineages

Hansen

Chamberlain

Trevena

et al. 2021

Genesis

Self Cite

View full text Add to dashboard Cite

Summary In many animal species, germ cell specification requires the inheritance of germ plasm, a biomolecular condensate containing maternally derived RNAs and proteins. Most studies of germ plasm composition and function have been performed in widely evolutionarily divergent model organisms, such as Caenorhabditis elegans, Drosophila, Xenopus laevis, and Danio rerio (zebrafish). In zebrafish, 12 RNAs localize to germ plasm at the furrows of the early embryo. Here, we tested for the presence of these RNAs in three additional species within the Danionin clade: Danio kyathit, Danio albolineatus, and Devario aequipinnatus. By visualizing nanos RNA, we find that germ plasm segregation patterns during early embryogenesis are conserved across these species. Ten additional germ plasm RNAs exhibit localization at the furrows of early embryos in all three non‐zebrafish Danionin species, consistent with germ plasm localization. One component of zebrafish germ plasm, ca15b, lacked specific localization in embryos of the more distantly related D. aequipinnatus. Our findings show that within a subset of closely related Danionin species, the vast majority of germ plasm RNA components are conserved. At the same time, the lack of ca15b localization in D. aequipinnatus germ plasm highlights the potential for the divergence of germ plasm composition across a restricted phylogenetic space.

show abstract

Section: Methodsmentioning

confidence: 99%

Conserved germ plasm characteristics across theDanioandDevariolineages

Hansen

Chamberlain

Trevena

et al. 2021

Genesis

Self Cite

View full text Add to dashboard Cite

show abstract

A maternally inherited Chromosomal Passenger Complex regulates germ plasm ribonucleoparticle aggregation in Zebrafish

Moravec,

Pelegri

2024

Preprint

View full text Add to dashboard Cite

In zebrafish, the formation of primordial germ cells depends on the inheritance of a compartmentalized membrane-less subcellular structure containing a pool of maternally expressed germ plasm ribonucleoparticles (gpRNPs) and proteins. Interactions between cytoskeletal components and gpRNPs are crucial for the movement and collection of gpRNPs into the furrows during the first few cellular division of the early embryo. Previous work has identified motley/ birc5b, a maternally-expressed homolog of a known Chromosomal Passenger Complex (CPC) component, Survivin, as a linker between gpRNPs and microtubules during gpRNP aggregation. However, Survivin can also function independent of the CPC in other cellular contexts. Here we investigated whether a maternally inherited CPC is necessary for gpRNP aggregation. We identified cdca9 as a maternally-expressed duplicated homolog of Borealin, another member of the CPC. Similar to motley, embryos from homozygous cdca9 mutant females exhibit defects in chromosome segregation and cytokinesis during meiosis and mitosis, phenotypes associated with mutations in CPC members. Additionally, embryos lacking Cdca9 displayed decreased gpRNP aggregation prior to furrow formation in the early embryo, a phenotype indistinguishable from that observed in motley mutants. As previously shown for Birc5b, Cdca9 and other CPC components INCENP and Aurora B kinase colocalize at the tips of astral microtubules as gpRNPs are transported to the forming furrow. Unexpectedly, Birc5b, but not other CPC components, accumulates within the growing gpRNP aggregate prior to and during furrow formation. The association of Birc5b with germ plasm masses continues during their asymmetric segregation in the cleavage stages, ceasing only when gpRNPs undergo cytoplasmic dispersal during gastrulation. Our studies reveal a role for a non-conventional, maternally-inherited CPC for spindle and furrow formation, and, unexpectedly, gpRNP aggregation during early development. Additionally, we find that Birc5b, but not other CPC proteins, remains a component of zebrafish germ plasm during and after its aggregation.

show abstract

Mechanical constraints organize 3D tissues and orchestrate muscle differentiation

Nagle,

van der Spek,

Gesenhues

et al. 2024

Preprint

View full text Add to dashboard Cite

Biological tissues achieve proper shape and ordered structures during development through responses to internal and external signals, with mechanical cues playing a crucial role. These forces guide cellular organization, leading to complex self-organizing structures that are foundational to embryonic patterns. Emerging theories and experiments suggest that "topological morphogens" drive these processes. Despite the predominance of three-dimensional (3D) structures in biology, studying 3D tissues remains challenging due to limited model systems and the complexity of modeling. Here, we address these challenges by using self-organized cellular aggregates, specifically spindle-shaped C2C12 myoblasts, subjected to controlled mechanical stretching. Our findings reveal that these cells form a multilayered, actin-oriented tissue structure, where mechanical forces drive long-range 3D organization and muscle differentiation. Notably, tissue surface emerges as a hotspot for differentiation, correlating with directional order as shown by single molecule fluorescent \textit{in situ} hybridization.

show abstract

Methods for Visualization of RNA and Cytoskeletal Elements in the Early Zebrafish Embryo

Cited by 4 publications

References 19 publications

Conserved germ plasm characteristics across theDanioandDevariolineages

Conserved germ plasm characteristics across theDanioandDevariolineages

A maternally inherited Chromosomal Passenger Complex regulates germ plasm ribonucleoparticle aggregation in Zebrafish

Mechanical constraints organize 3D tissues and orchestrate muscle differentiation

Contact Info

Product

Resources

About