Essential and dual effects of Notch activity on a natural transdifferentiation event

Abstract

Cell identity can be reprogrammed, naturally or experimentally, albeit with low frequency. Why some cells, but not their neighbours, undergo a cell identity conversion remains unclear. We find that Notch signalling plays a key role to promote natural transdifferentiation in C. elegans hermaphrodites. Endogenous Notch signalling endows a cell with the competence to transdifferentiate by promoting plasticity factors expression (hlh-16/Olig and sem-4/Sall). Strikingly, ectopic Notch can trigger additional transdifferentiation in vivo. However, Notch signalling can both promote and block transdifferentiation depending on its activation timing. Notch only promotes transdifferentiation during an early precise window of opportunity and signal duration must be tightly controlled in time. Our findings emphasise the importance of temporality and dynamics of the underlying molecular events preceding the initiation of natural cell reprogramming. Finally, our results support a model where both an extrinsic signal and the intrinsic cellular context combine to empower a cell with the competence to transdifferentiate.

Fig. 1. Ectopic Notch activity during early-embryogenesis results in an extra transdifferentiation event.

A Timeline of the Y-to-PDA Td. The rectum of the worm is composed of 6 specialised epithelial cells (Y, B, U, F, K and K′) arranged in three rings. These cells, as well as DA9^prog, are born in the embryo around 290 min after the first cleavage and are fully differentiated when the worm hatches. The Y cell keeps its epithelial identity until the end of the first larval stage, when Td is initiated. Y retracts from the rectum and migrates antero-dorsally, while it is replaced by P12.pa. Concomitantly, the Y identity is erased in a sensu strictu dedifferentiation step. Later in L2, re-differentiation into a motoneuron begins, to adopt the “PDA” final identity by the L3 stage. This sterotyped event is identifiable and predictable in all WT animals. B Quantification (in %) of [2 PDA] phenotype in WT, strong lin-12^Notch(n950) and mild lin-12^Notch(n302) gain-of-function alleles. C Quantification (in %) of [0 DA9; 2 PDA] in lin-12^Notch(n950). D Top panel: Lineage tree and fate of DA9^prog and Y^prog cells in WT, and lin-12^Notch(gf) animals. Dotted line represents cell division not displayed in the scheme (after Sulston et al. 1983). Bottom panel: Pictures of the WT (left) and lin-12^Notch(n950) [0 DA9; 2 PDA] (right) worms. White arrowheads: PDA neuron. Magenta arrowheads: DA9 neuron. Scale bar: 10 µM. E Quantification (in %) of the number of PDA and DA9 neurons found in a lin-12^Notchp::NICD::lin-12^Notch3UTR′ transgenic line. F Quantification (in %) of worms exhibiting [2 PDA] in independent transgenic lines expressing the hsp::NICD construct, after a heat shock during early-embryogenesis (see HS 1, Fig. 2C). B–F n, total number of animals scored. Tg, transgenic animals. Non-Tg non-transgenic (control) siblings. cog-1::gfp, PDA marker. mig-13p::mCherry or itr-1p::mCherry: DA9 markers. %, proportion of worms showing the mutant phenotype over all worms scored (ie, penetrance). Data represent the mean of at least three biological replicates (dots represent the mean of each replicate). Two-tailed P value (in grey) is calculated using a Chi² test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 2. lin-12^Notch receptor is expressed from early to mid-embryogenesis.

A Expression pattern and dynamics of lin-12^Notchp::gfp (rtEx727). lin-12^Notch is expressed in Y (arrowhead) during embryogenesis from its birth to the twofold stage. In the threefold stage, out-of-focus background GFP signal is due to expression of myo-2p::gfp, the co-injection marker used to generate this transgenic line; no lin-12^Notchp::gfp signal is detected in Y nor DA9 at this stage. B Expression pattern and dynamics of the LIN-12^Notch protein (arIs41). LIN-12^Notch is present in Y during embryogenesis from Y birth to the twofold stage (where a faint signal can still be detected). A, B Dotted line, rectal slit. Anterior is to the left and ventral to the bottom. Inserts represent blown-up rectal areas. White arrowheads: Y cell. Magenta arrowheads: DA9 neuron. Numbers represent the fraction of worms displaying this representative expression pattern over the total number of worms scored. N = 1. Scale bar: 10 µM. C Timing of expression of the lin-12, egl-5 and col-34 genes or drivers used, and of the heat-shocks (HS) 1, 2 and 3 with respect to the timeline of the different steps of the Y-to-PDA Td.

Fig. 3. hlh-16 is necessary for Y Td.

A Quantification (in %) of PDA presence at the L4 stage (after Td) in hlh-16(fp12). cog-1::gfp, PDA marker. B Quantification (in %) of a persistent Y cell in the rectum at the L4 stage (after Td) in hlh-16(fp12), assessed with egl-5 reporter. A, B n, total number of animals scored. Data represent the mean of replicates (dots: mean of each replicate). %, proportion of worms showing the mutant phenotype over all worms scored (i.e., penetrance). Two-tailed P value (in grey) is calculated using a Chi² test. ****P < 0.0001. C Picture of the phenotypes represented in (B). Top: WT L4 animals; bottom: hlh-16(fp12) L4 mutant. White arrowhead: absence of Y in WT. Magenta arrowhead: persistent Y cell in the rectum. Scale bar: 10 µM. D HLH-16::GFP is expressed in Y^prog but not in DA9^prog in the early WT embryo (1.5-fold). Insert represents blown-up rectal area. Dotted line, rectal slit. Numbers represent the fraction of worms displaying this representative expression pattern over the total number of worms scored. N = 1. Scale bar: 10 µM.

Fig. 4. Additional Td requires endogenous Td factors, unveiling hlh-16 and sem-4 as downstream of Notch.

A Plasticity factors necessary for the WT Y-to-PDA Td are also required for the extra Td ([2 PDA]) in lin-12^Notch(gf). The [2 PDA] (in %) is significantly suppressed when the plasticity factors (sem-4, egl-27, ceh-6, sox-2, egl-5 and hlh-16) are knocked-down by RNAi in lin-12^Notch(n950) and a concommittant 0 PDA phenotype is observed (dpy-8, negative control). See also Supplementary Fig. 2A, B. B Significant suppression of the lin-12^Notch(gf) [2 PDA] (in %) when unc-3 is knocked down by RNAi in lin-12^Notch(n950). dpy-8, negative control. Of note, unc-3(RNAi) is poorly efficient and leads to a very low penetrance defect. A, B cog-1::gfp, PDA marker. itr-1p::mCherry, DA9 marker. Quantification of the number of HLH-16 + Y (C) and SEM-4 + Y (F) cells in the rectum (in %) of lin-12^Notchp::NICD L1 animals. HLH-16::GFP (D) and sem-4p::GFP (G) are expressed in Y WT L1 larva (white arrowhead). As quantified in (C, F), 2 Y cells expressing HLH-16::GFP (magenta and white arrowheads, D) and sem-4p::GFP (G) are found in lin-12^Notchp::NICD. E sem-4p::gfp is expressed in Y^prog but not in DA9^prog in the late WT embryo (threefold). N = 1. Numbers in bottom left corner represent the fraction of worms displaying this representative expression pattern over the total number of worms scored. A–C, F) n, total number of animals scored. %, penetrance of the mutant phenotype shown. Two-tailed P value (in grey) is calculated using a Chi² test. Tg: transgenic; non-Tg: non-transgenic (control) siblings. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. D, E, G Anterior to the left, and ventral at bottom. Dotted line: rectal slit. Scale bar: 10 µM.

Fig. 5. Ectopic Notch signal in rectal cells is not sufficient to form additional PDAs.

Left, experimental approach: overexpression of the NICD construct in all the rectal cells using the egl-5 promoter. Representative pictures of the specified marker expression in a L4 worm (A, C) or a L3 worm (B), showing that the rectal identity of the B, U, F, K and K′ rectal cells is not affected by NICD overexpression. SL2::mCherry shows NICD expression in the nucleus of rectal cells (see also Supplementary Fig. 3). No constipation is observed, showing that these rectal cells are fully functional. Bottom left corner, number of animals showing the represented phenotype over the total number of animals scored. N = 2. Anterior is to the left and ventral to the bottom. Arrowheads show the position of the designated cells. Dotted line, rectal slit. Scale bar: 10 µM. D–E High penetrance of Td defect [0 PDA] (in %) is found in transgenic (“Tg”) worms expressing egl-5p::NICD::SL2mCherry (D) or col-34p::NICDGFP (E) in five independent transgenic lines. No extra Td [2 PDA] was observed. Non-transgenic (“Non-Tg”) control siblings are WT as observed with cog-1::gfp reporter. See Fig. 2C for the timing of egl-5 and col-34 expression. F, G Quantification (in %) of worms exhibiting an extra Td [2 PDA] or no Td [0 PDA] phenotype after a heat shock during (F) mid-embryogenesis (HS 2, Fig. 2C) and (G) before Td initiation in late L1 stage (HS 3, Fig. 2C), in three independent transgenic lines expressing the hsp::NICD construct, as assessed with cog-1::gfp reporter. D–G n, total number of animals scored. %, penetrance of the mutant phenotype shown. Data represent the mean of 3 replicates; dots: mean of each replicate. Two-tailed P value, indicated in grey, is calculated using a Chi² test. ****P. < 0.0001, ***P. < 0.001, **P. < 0.01, *P. < 0.05.

Fig. 6. A late Notch signal cell-autonomously blocks initiation of Td.

A Experimental approach: overexpression of the NICD construct in a subset of the rectal cells using the lin-48 promoter. B No Td defects ([0 PDA], in %) is observed when NICD (lin-48p::NICD::SL2mCherry) is expressed in the F, U, K and K′ rectal cells in five independent transgenic lines as assessed using cog-1::gfp reporter. Tg transgenic worms, Non-Tg non-transgenic WT siblings, n total number of worms scored. Data represent the mean of 3 replicates; dots: mean of each replicate. A similar proportion (C) or even a higher proportion (D) of animals (in %) exhibit a Td defect [0 PDA] when NICD is not expressed (white bar) in the B cell using a mosaic egl-5 driver (C) or a mosaic egl-20 driver (D), revealing no correlation between Td defect and expression in the B cell; n, total number of animals scored = 61 (C) and 51 (D). E Representative DIC picture of the persistent Y cell, and thus ([0 PDA]) as quantified in (C), in L4 transgenic animals expressing ectopic activated Notch in the rectal cells (egl-5p::NICD). Left, WT worm where Y has turned into a PDA (star). Right, L4 worm with ectopic Notch activation in Y. The Y cell (arrowhead) is blocked before Td initiation and found at its original position in the rectum; the rectal U and P12.pa cells (arrowheads) are also visible. Dotted line, rectal slit. Anterior is right and ventral is to the bottom. Scale bar: 10 µM. F The persistent Y cell remains rectal. Epithelial and neuronal markers in the persistent Y cell (expressed in number of animals) in worms expressing an integrated NICD construct under the egl-5 promoter (egl-5p::NICD). When Y Td is blocked, a persistent Y cell is found at its original position (160/200 animals, N = 4). This Y cell remains rectal. See also Supplementary Fig. 4. n, total number of animals scored.

Fig. 7. Regulation of Notch signal duration is achieved through transcriptional regulation.

A–D lag-2 and apx-1 act as ligands for activation of Notch in the Y cell. Permissive temperature, 15 °C; restrictive temperature, 26.5 °C. A) Quantification (in %) of [0 PDA] phenotype in lag-2(q420ts) mutant. B Quantification (in %) of [0 PDA] in single apx-1(zu347ts) and double apx-1(zu347ts); sel-12(ar171) mutants. Notch pathway mutant sel-12(ar171) does not exhibit Td defect and is thus used as a sensitised background, see also Supplementary Fig. 5A. C lag-2 is expressed in B (arrowheads) from Y birth until Td initiation. N = 1. Scale bar: 10 µM. D apx-1 is expressed in four cells (arrowheads) close to the rectum during embryogenesis. C, D Dotted line: rectal slit. Inserts represent blown-up rectal areas. Numbers represent the fraction of worms displaying this representative expression pattern over the total number of worms scored. N = 1. Scale bar: 10 µM. E Ligands are still available and functional when lin-12^Notch is down-regulated. Last 2 bars, proportion of [0 PDA] worms carrying a col-34p::lin-12^Notch(WT)cDNA transgene. Negative control transgenic lines: inactive LIN-12^Notch receptors driven by col-34p [col-34p::lin-12^NotchcDNA(n941) and col-34p::lin-12^NotchcDNA(∆ANK)]. Positive control: constitutively active full-length receptor driven by col-34p [col-34p::lin-12^NotchcDNA(n137)]. F Quantification (in %) of the number of PDA and DA9 neurons found in transgenic lines expressing NICD under different lin-12^Notch promoter variants. First 2 bars: lin-12^Notchp::NICD transgenic line (same data as Fig. 1E). Middle 2 bars: lin-12^Notchp(ΔR1)::NICD line. Right-most 2 bars: lin-12^Notchp(ΔR2)::NICDGFP line. G The lin-12^Notch promoter region (black rectangle) used to drive NICD expression. Light green and green tracks represent the overall conservation of the DNA sequence between different Caenorhabditis species and C. elegans respectively (adapted from UCSC genome browser). The red boxes identify two conserved regions (R1, R2) of the lin-12^Notch promoter further tested. A, B, E, F) PDA marker: cog-1::gfp. DA9 marker: itr-1p::mCherry. Tg transgenic worms, non-Tg non-transgenic siblings. n total number of animals scored. %, penetrance of the mutant phenotype shown. Data represent the mean of replicates; dots: mean of each replicate. Two-tailed P value, indicated in grey, is calculated using a Chi² test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Additional lines are depicted in Supplementary Fig. 6A–G.

Fig. 8. Notch level regulates the cell-fate decision between DA9 and Y/PDA fates.

A Quantification (in %) of PDA and DA9 assessed with respectively cog-1 and itr-1 reporters in lin-12^Notch(n676n930) at 15 °C. B Quantification (in %) of Y cell in lin-12^Notch(n676n930) at 15 °C assessed with egl-5 reporter at early L1 stage (left) and early L2 stage (right). [1Y], WT Y cell (without any axon). C Quantification (in %) of Y and DA9 cell assessed with respectively hlh-16 and mig-13 reporters in lin-12^Notch(n676n930) at 15 °C at early L1 stage (left) and early L2 stage (right). D Representative picture of [1Y; 1DA9] WT phenotype and [0Y; 2DA9] observed in lin-12^Notch(n676n930) L1 animals at 15 °C, assessed with respectively hlh-16 (Y::GFP) and mig-13 (DA9::mCherry) reporters. E Quantification (in %) of Y cell expressing endogenously tagged ngn-1(dev137) allele in lin-12^Notch(n676n930) at 15 °C at early L1 stage (left) and early L2 stage (right). Quantification (in %) of Y cell and DA9 neuron in lin-12^Notch(n676n930), assessed with respectively ngn-1 reporter from Rashid et al. 2022 (nsIs913) and mig-13 reporters at 15 °C (F) at early L1 stage (left) and early L2 stage (right), and at 25 °C (G). Except for 3/92 [1DA9; ngn-1+ “neuronal”] animals (F), all the cells expressing nsIs913 always also expressed the DA9 marker. H Picture of [1Y; 1DA9] phenotype in WT (top) and [0Y; 2DA9] observed in lin-12^Notch(n676n930) (bottom) early L1 at 15 °C assessed with respectively ngn-1 reporter (nsIs913) from Rashid et al. and mig-13 reporters. A–C, E–G n total number of animals scored. %, penetrance of the mutant phenotype shown. Data represent the mean of replicates (dots: mean of each replicate). Two-tailed P value, indicated in grey, is calculated using a Chi² test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. D, H Dotted line, rectal slit; white arrowhead, Y cell; magenta arrowhead, DA9 neurons. The rectal area is pictured. Anterior is to the left and ventral to the bottom. Scale bar: 10 µM.

Fig. 9. Model for the impact of Notch activity on natural Y-to-PDA.

In the WT situation, a pulse of Notch triggered by two redundant ligands (apx-1 and lag-2) around Y birth will activate two potential sets of genes, endowing the Y cell with its rectal epithelial identity as well as its competence to transdifferentiate. The action of these 2 sets can be distinguished, as at least two genes downstream of Notch are crucial for the initiation of Td while not impacting on Y identity. This time interval of Notch signal represents the window of opportunity during which the Notch pathway can promote Td. In WT animals, Notch signal is then down-regulated at the transcriptional level to allow the Y cell to initiate Td, the first step being the erasure of its initial identity. When a later Notch signal is provided to the Y cell, it does not reset a timer and delay Td, but results in a block of Td by over-imposing in Y a rectal epithelial fate. Together, it shows how one signalling pathway, Notch, can exert opposite effects if activated at different steps, on Y-to-PDA Td, a step-wise process that occurs in the absence of cell division. It further shows that both the extrinsic environment, as well as the intrinsic cellular context, combine to allow one cell to switch identity.