Thambyrajah et al. · Nature Communications · 2024

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate

Thambyrajah Roshana, Maqueda Maria, Neo Wen Hao, Imbach Kathleen, Guillén Yolanda, Grases Daniela, Fadlullah Zaki, Gambera Stefano, Matteini Francesca, Wang Xiaonan, Calero-Nieto Fernando J., Esteller Manel, Florian Maria Carolina, Porta Eduard, Benedito Rui, Göttgens Berthold, Lacaud Georges, Espinosa Lluis, Bigas Anna

Read the paper The product used

The study

What was asked, and what was found

Thambyrajah et al., Nature Communications, 2024, asked how the first blood stem cells in a mouse embryo keep their identity while Notch signalling is going on all around them. The answer they found is that the receptor NOTCH1 and the ligand JAG1 sit on the surface of the same cell rather than signalling between neighbours. Proximity ligation assays reading the intracellular tails of both proteins showed that pairing directly, and single cell sequencing showed the glycosyltransferase RFNG present in exactly the stem cell population where the pairing matters.

The experiment that closes the argument used an AUMsilence sdASO. Explants of the mouse embryonic aorta, gonads and mesonephros were taken at embryonic day 10.5 and cultured for 2 days with either an oligo designed against the messenger RNA of Rfng or a matched scramble control, added to the culture medium with no transfection reagent used at any stage. The authors describe the oligo entering the cells and hybridizing to the transcript, which RNase H then degrades, and they confirmed the knockdown by measuring RFNG protein by flow cytometry.

After 48 hours the knockdown explants had significantly fewer cells carrying the stem cell surface phenotype and a reduced fraction co-expressing NOTCH1 and JAG1, across 3 independent experiments and 24 embryos. Removing the enzyme therefore removed the pairing, and removing the pairing cost the tissue its stem cells. What makes the experiment worth noting for anyone choosing a reagent is the preparation: a piece of intact embryonic tissue in culture, not a cell line, silenced by adding the oligo to the medium. One caution belongs with any use of this paper. The figure legend and the Methods give the concentration as 10 μM and 10 nM respectively, and the paper never reconciles them.

Key findings

An AUMsilence sdASO against Rfng, added to mouse embryonic aorta explants for 2 days, left the tissue with significantly fewer stem cells by surface phenotype and with less NOTCH1 and JAG1 co-expression than the scramble control.(Results, page 9, Fig. 7F and 7G)
That knockdown is what moved the paper from correlation towards mechanism: with the glycosyltransferase knocked down, stem cell numbers fell and the receptor and ligand pairing on the same cell was reduced, which the authors read as the glycosyltransferase favouring that pairing and the pairing being needed to keep a stem cell a stem cell.(Results, page 9)
The oligo reached cells inside an intact piece of embryonic tissue in culture, with no transfection reagent used at any point, and the authors describe it degrading the target transcript through RNase H.(Results, page 9)
The wider study found that the receptor and the ligand sit together on the surface of the same stem cell rather than signalling between neighbours, which is the arrangement the knockdown then tested.(Discussion, page 9)

Product: AUMsilence sdASO
Targets: Rfng · mouse · mRNA
Models: mouse embryonic aorta, gonads and mesonephros explants from embryonic day 10.5, cultured 2 days · ex vivo tissuethe same explants read out by flow cytometry 48 hours after the oligo was added · ex vivo tissue
Delivery: added to the explant culture medium for the whole 2 day culture, with no transfection reagent named anywhere in the paper
Readouts: flow cytometry for RFNG protein and for the stem cell surface markers, immunohistochemistry, proximity ligation assay, single cell RNA sequencing with index sorting, nascent RNA capture and sequencing, quantitative PCR
Measured: oligo concentration as the figure legend gives it: 10 μM (both the scramble and the targeting oligo, in the explant culture, 48 h)oligo concentration as the Methods give it, which contradicts the figure legend by a factor of a thousand: 10 nM (both the scramble and the targeting oligo, during the culture period)effect of the knockdown on stem cells and on the receptor and ligand pairing: significantly fewer phenotypic stem cells and reduced NOTCH1 and JAG1 co-expression (48 h of explant culture, against the scramble oligo)replication behind the knockdown result: 3 independent experiments, 24 embryos (two-tailed t tests on the frequency of cKIT, cKIT and CD45 double positive, and the T2 stem cell gate)how the knockdown itself was confirmed: RFNG protein by flow cytometry, no numeric knockdown efficiency reported anywhere in the paper (gated on CD31 and cKIT double positive cells, and again on CD45)
Figures: Fig. 7 Where the glycosyltransferase sits in the embryonic aorta, and what happens to the stem cell gate and to receptor and ligand co-expression when it is knocked down in explants for 48 hours against a scramble oligo.Fig. 7F The explant experiment schematic and the flow plots for the stem cell markers in scramble against knockdown explants.Fig. 7G Quantification across 3 experiments and 24 embryos of the cKIT, cKIT with CD45, and T2 stem cell gates in scramble against knockdown explants.Supplementary Fig. 7B The flow cytometry check that the knockdown lowered RFNG protein in the explants.
Citation: Thambyrajah et al. Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate. Nature Communications 2024;15:1604. doi:10.1038/s41467-024-45716-y