Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

Lu, Yang; Titlow, Josh; Ennis, Darragh; Smith, Carlas; Mitchell, Jennifer; Young, Florence L.; Waddell, Scott; Ish‐Horowicz, David; Davis, Ilan

doi:10.1016/j.ymeth.2017.06.025

Cited by 47 publications

(36 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, we found that the expression peaks of four marker genes, i.e. CycE for INPs (Yang et al, 2017), dap for GMCs (Lane et al, 1996;de Nooij et al, 1996), Hey for a subset of the transient immature neuronal state (Monastirioti et al, 2010), and nSyb for maturing neurons (Deitcher et al, 1998), aligned in this exact differentiation order along the calculated pseudotimeline ( Fig. 2C).…”

Section: Pseudotime Analysis Describes the Continuous Differentiationmentioning

confidence: 78%

“…Cells expressing high levels of the INP markers CycE and D are good candidate starting cells for Palantir (Bayraktar and Doe, 2013;Yang et al, 2017). To easily identify these cells from the UMAP plot, we built a Multi-informatic Cellular Visualization web tool (MiCV) to allow displaying the single cell co-expression pattern of multiple genes in the 2D/3D UMAP plots.…”

Section: Pseudotime Analysis Describes the Continuous Differentiationmentioning

confidence: 99%

See 1 more Smart Citation

The molecular landscape of neural differentiation in the developingDrosophilabrain revealed by targeted scRNA-seq and a multi-informatic analysis paradigm

Michki

Sanjasaz

et al. 2020

Preprint

View full text Add to dashboard Cite

SUMMARYTo adopt terminal neural fates, neural progenitors respond to a combination of molecular factors, the interplay between which remains difficult to untangle. In this work, we interrogated the type-II neuroblast lineages in the developing Drosophila brain using targeted single-cell mRNA sequencing. We used pseudotime analysis to group cells by their differentiation state and subsequently used marker gene analysis to identify genes that may play a role in neural fate specification. To aid the candidate gene selection process, we created MiCV, a scRNA-seq data visualization web tool that integrates results from the multifaceted analysis, displays the co-expression pattern of multiple genes, and provides an automated gene function curating feature. In situ profiling of these mRNAs further recovered the spatial information lacking in the single-cell data. Combining prior knowledge, in silico analysis, and in situ evidence, we characterize the molecular landscape of the developing Drosophila type-II neuroblast lineages and provide a roadmap for navigating the more complex brains.

show abstract

Section: Pseudotime Analysis Describes the Continuous Differentiationmentioning

confidence: 78%

Section: Pseudotime Analysis Describes the Continuous Differentiationmentioning

confidence: 99%

The molecular landscape of neural differentiation in the developingDrosophilabrain revealed by targeted scRNA-seq and a multi-informatic analysis paradigm

Michki

Sanjasaz

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…For in-situ hybridisation experiments, RNA probes were designed using LGC Biosearch Technologies’ Stellaris® RNA FISH Probe Designer against Cap-G exon 4 and with Quasar® 570 dye. Tissue was treated following the published protocol [74]. Fixed and blocked tissue was incubated in hybridisation buffer with Cap-G probe (3μM) and primary antibody of interest for 8-15 hours.…”

Section: Methodsmentioning

confidence: 99%

Condensin I subunit Cap-G is essential for proper gene expression during the maturation of post-mitotic neurons

Hassan

Rodriguez

Heidmann

et al. 2020

Preprint

View full text Add to dashboard Cite

AbstractThe condensin complex is essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about its function in post-mitotic, differentiated cells. Here we report a novel role for the condensin I subunit Cap-G in Drosophila neurons. We show that, despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G and that knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes. These include reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in more mature neurons also results in similar phenotypes but to a lesser degree. Furthermore, we see dynamic binding of Cap-G to chromatin at distinct loci in neural stem cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.

show abstract

“…Pros protein is expressed at low levels in larval type I NBs and GMCs, where it promotes GMC differentiation, and is then upregulated in larval neurons (Carney et al, 2013;Choksi et al, 2006). To determine whether the upregulated Pros expression in neurons is driven by an increase in pros mRNA levels or by increased Pros translation, we used smFISH in whole-mount Drosophila larval brains at the wandering third instar stage (Yang et al, 2017b). We carried out four colour imaging with smFISH against pros exon ( Figure 1A), anti-Pros antibody, anti-Elav antibody (marking differentiated neurons) and DAPI (marking DNA in all cells) ( Figure 1B).…”

Section: Upregulation Of Pros Protein In Neurons Is Achieved Throughmentioning

confidence: 99%

Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation

Samuels

Arava

Järvelin

et al. 2017

Preprint

Self Cite

View full text Add to dashboard Cite

During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled.Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long prospero isoform, which allows an upregulation of Prospero protein production. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.

show abstract

Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

Cited by 47 publications

References 39 publications

The molecular landscape of neural differentiation in the developingDrosophilabrain revealed by targeted scRNA-seq and a multi-informatic analysis paradigm

The molecular landscape of neural differentiation in the developingDrosophilabrain revealed by targeted scRNA-seq and a multi-informatic analysis paradigm

Condensin I subunit Cap-G is essential for proper gene expression during the maturation of post-mitotic neurons

Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation

Contact Info

Product

Resources

About