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. 2018 Feb 8;172(4):869-880.e19.
doi: 10.1016/j.cell.2018.01.002. Epub 2018 Feb 1.

Dynamic Ligand Discrimination in the Notch Signaling Pathway

Nagarajan Nandagopal  1 , Leah A Santat  1 , Lauren LeBon  2 , David Sprinzak  3 , Marianne E Bronner  4 , Michael B Elowitz  5
Affiliations

Dynamic Ligand Discrimination in the Notch Signaling Pathway

Nagarajan Nandagopal et al. Cell. .

Abstract

The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes.

Keywords: Notch pathway; intercellular signaling; ligand multiplicity; myogenesis; signal decoding; signal encoding; signaling dynamics; single cell dynamics; systems biology.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Dll1 and Dll4 Activate Notch1 with Pulsatile and Sustained Dynamics, Respectively
(A) Both Dll1 (blue) and Dll4 (red) activate the Notch1 receptor (green) to induce proteolytic release of the Notch intracellular domain (NICD), but are used in different biological contexts (blue and red boxes, bottom). The released NICD translocates to the nucleus and, in complex with CSL/RBPjκ (yellow), activates Notch target genes (white). (B) Left: Engineered CHO-K1 “sender” cell lines contain stably integrated constructs expressing Dll1 (blue) or Dll4 (red), each with a co-translational (T2A, brown) H2B-mCh readout (purple), from a 4epi-Tetracycline (4epi-Tc) inducible promoter. Right: “Receiver” cells stably express a chimeric receptor combining the Notch1 extracellular domain (Notch1ECD) with a Gal4 transcription factor (orange), which can activate a stably integrated fluorescent H2B-3xCitrine reporter gene (chartreuse). (C) Left (schematics): A minority of receiver cells (green) are co-cultured with an excess of either Dll1 (blue) or Dll4 (red) sender cells. Right: Filmstrips showing representative sustained (top, Dll4 senders) or pulsatile (bottom, Dll1 senders) response of a single receiver cell (center, automatically segmented nucleus outlined in white). Grey channel shows DIC images of cells, while the rate of increase in Citrine fluorescence, scaled to 25%–75% of its total range, is indicated using green pseudo-coloring. See also Movies S1 and S2. (D) Left: Representative traces showing total nuclear Citrine fluorescence levels (top) or corresponding derivatives of the total Citrine (dCitrine/dt), i.e., promoter activity (bottom), in individual receiver cells activated by Dll4. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll4. Solid traces represent medians, lighter shades indicate SEM, and gray shading indicates SD. n, number of traces included in the alignment. See STAR Methods for alignment and normalization procedure. (E) Left: Corresponding plots (as in D) showing total nuclear Citrine fluorescence levels (top) and promoter activity (bottom) in individual receiver cells in co-culture with Dll1. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll1. The percentage value (60%) in the plots on right indicates the fraction of receiver traces included in the alignment (STAR Methods, see also Figure S1F). (F) 95th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in the traces included in (D) and (E). This time window is chosen to simultaneously estimate the promoter activity at the peak of Dll1 pulses and at steady-state levels of Dll4 signaling. Solid horizontal lines represent medians, while the boxes delineate 25th–75th percentile values. p value calculated by two-sided Kolmogorov-Smirnov (K-S) test. See also Figures S1 and S2.
Figure 2.
Figure 2.. Differences in Dll1 and Dll4 Dynamics Are Preserved across a Range of Ligand Expression Levels, and Ligand-Levels Modulate These Dynamics in Different Ways
(A) Left: Schematic of co-culture assay showing Dll1 (blue) or Dll4 (red) sender cells surrounded by receiver cells (green). Right: Filmstrips showing sustained or pulsatile responses in a single receiver cell (green, automatically segmented nucleus outlined in white) neighboring either Dll4 (top, nuclei pseudo-colored in red) or Dll1 (bottom, nuclei pseudo-colored in blue) sender cells. The gray channel shows DIC images, in which other receiver cells can be seen. Intensity of green in the receiver cell indicates promoter activity scaled to 25%–75% of its range. See also Movies S4 and S5. (B) Median response profiles in individual receiver cells co-cultured with sender cells expressing low, medium, or high levels of Dll4 (left) or Dll1 (right). See Figures S2J and S2K for ligand expression levels in each group. Solid traces represent medians, light colored regions indicate SEM, gray shading indicates SD. n values indicate number of receiver cell responses included in the alignment. The percentage values in the Dll1 plots indicate the fraction of receiver traces included in the alignment (STAR Methods). (C) Left: Comparison of maximal promoter activities (95th percentile of promoter activity values in each trace) in activated receiver cells adjacent to sender cells expressing no ligand (black), or low (red), medium (pink), or high (dark red) levels of Dll4 (same designations as used in B). Right: Similar comparison for Dll1. Grey circles represent individual responses, solid horizontal lines represent medians, while the boxes delineate 25th–75th percentile values. p values calculated by two-sided K-S test. Not significant (ns), p > 0.01. (D) Median values of the number of receiver cells activated by isolated Dll1 sender cells expressing low, medium, or high levels of co-translational H2B-mCherry and their progeny during a 25 hr experiment under excess receiver conditions. Error bars represent SEM. (E) Schematic: Summary of Dll1 and Dll4 modulation. Dll1 levels primarily control rate or frequency of stereotyped pulses, while Dll4 levels control amplitude of sustained signal. See also Figures S2J–S2L and Movie S3.
Figure 3.
Figure 3.. Pulsatile and Sustained Notch Activation Can Regulate Different Sets of Target Genes
(A) C2C12 cells were engineered to expressed Notch1 receptors lacking the extracellular domain (N1DECD, green). This receptor is inactive in the presence of the γ-secretase inhibitor DAPT (red), but constitutively active when DAPT concentration is reduced in the culture medium. (B) Comparison of transcript levels in C2C12-N1ΔECD cells at 1 hr or 6 hr after DAPT removal. The blue line represents equal expression at 1 hr and 6 hr, and the gray lines represent 5-fold changes in either direction. Circled genes are putative direct Notch targets. The blue circle highlights target genes that are upregulated >5-fold at 1 hr but not 6 hr, while red circles indicate target genes that are upregulated >5-fold only after 6 hr. See also Figure S3 and Table S1. (C) qPCR time course measurement of Hes1 (blue), Hey1 (orange), and HeyL (yellow) mRNA levels following complete DAPT removal at t = 0 hr. (D) Duration dependence of Hes1 (blue) and Hey1 (orange) response to DAPT removal for 5 min, 15 min, or 30 min followed by replenishment (“Pulse”), or no replenishment until the 1 hr or 4 hr measurement (“Sustained”). Error bars represent SEM calculated from duplicate experiments (n = 2). See also Figure S4.
Figure 4.
Figure 4.. Dll1 Expression in the Chick Neural Crest Promotes Myogenesis but Dll4 Inhibits It
(A) Developing chick embryo (dorsal view schematic). Dll1 (blue cells in 3) is expressed in a fraction of neural crest cells (gray, see 2, 3). These cells activate Notch1-expressing Pax7+ progenitor cells in the dorsomedial lip (DML, magenta) of the somite. When activated, these progenitor cells (green, 3) upregulate Hes1 and the muscle regulatory gene MyoD1. (B–D) Representative images showing effects of Dll1 or Dll4 electroporation into the neural crest, on Hes1, Hey1, and MyoD1 expression in the DML. White arrows indicate the somites on the electroporated side. The dotted lines indicate the DMLs of somites or the central line of the neural tube. (B) Top: Dll1-T2A-EGFP (i, blue), electroporated into the left side of the neural tube, is expressed in the neural tube and neural crest, resulting in upregulation of Hes1 (ii, red) and MyoD1 (iii, green) in the somites on the electroporated (left) side compared to the right side, which serves as negative control. Bottom: When Dll4-T2A-EGFP (iv, blue) is electroporated, Hey1 (v, red) is upregulated on the electroporated side, and MyoD1 (vi, green) expression is decreased. (C) Dll1-T2A-EGFP (blue, left) electroporation does not affect expression of Hey1 (red, right) in adjacent somites. (D) Dll4-T2A-EGFP (blue, left) electroporation increases expression of Hes1 (red, right) in adjacent somites. See also Table 1 and Figure S5.
Figure 5.
Figure 5.. Ligand Intracellular Domains Control Dynamic Signaling Mode and Influence Transendocytosis Patterns
(A and B) Dll4ECD-Dll1ICD and Dll1ECD-Dll4ICD were constructed by exchanging the intracellular domain (ICD) of Dll4 with that of Dll1. (A) Median response profiles in activated receiver cells co-cultured with Dll4 sender cells (red, top left) or Dll4ECD-Dll1ICD sender cells (magenta, right) under excess receiver conditions (as in Figure 2). Solid traces represent medians, lighter colored regions represent SEM, and gray shading represents SD. n, number of cell traces included in the alignment. See STAR Methods for alignment and normalization procedures. Bottom left: 95th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in individual traces included in the averaging. Solid horizontal lines represent medians, while the boxes delineate 25th–75th percentile values. p value calculated by two-sided K-S test. (B) Corresponding response profiles (right, top left) and amplitudes (bottom left) in activated receiver cells co-cultured with Dll1 sender cells (blue) or Dll1ECD-Dll4ICD sender cells (purple) under excess sender conditions. (C) Representative images of “excess sender” co-cultures of receiver cells (R) expressing full-length Notch1 and sender cells (S) expressing either Dll4ECD-Dll1ICD (left) or Dll4 (Dll4ECD-Dll4ICD, right), immunostained for Notch1ECD. Examples of dispersed, low intensity staining or higher-intensity puncta are indicated by the white circles. (D) Left: Median values of number of puncta detected (see STAR Methods) in Dll1ICD (blue) or Dll4ICD (red) sender cells neighboring receiver cells. Right: Median values of the (background subtracted) mean pixel intensity of dispersed signal (see STAR Methods) within Dll1ICD (blue) or Dll4ICD (red) sender cells that neighbor receiver cells. Error bars represent SEM. p value calculated using the two-sided K-S test. (E) Schematic: Proposed differences in the abilities of ligands containing the Dll1 (blue) and Dll4 (red) ICDs to initiate transendocytosis in different clustering states. See also Figure S6.
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