Notch exhibits ligand bias and maneuvers stage-specific steering of neural differentiation in embryonic stem cells

Abstract

Notch dictates multiple developmental events, including stem cell maintenance and differentiation, through intercellular communication. However, its temporal influence during early development and, of particular interest, its regulation of binary fate decision at different stages during neurogenesis are among the least explored. Here, using an embryonic stem cell (ESC) model, we have deciphered Notch ligand preference during ESC commitment to different germ layers and determined the stage-specific temporal effect of Notch during neural differentiation. ESCs during maintenance remain impervious to Notch inhibition. However, Notch activation promotes differentiation even in the presence of leukemia inhibitory factor (LIF), displaying ligand preference-associated lineage discrimination, where Jagged-1 favors neural commitment and Delta-like-4 favors the mesoderm. This differential ligand action involves a combination of Notch receptors influencing specific downstream target gene expression. Though Notch activation during early neural differentiation specifically promotes neural stem cells or early neural progenitors and delays their maturation, its inhibition promotes late neural progenitors and expedites neurogenesis, with a preference for neurons over glia. However, gliogenesis is promoted upon Notch activation only when executed in combination with ciliary neurotrophic factor. Thus, our investigation underscores a multifaceted role of Notch, demonstrating the interdependency of ligand usage and lineage specification and Notch acting as a master switch, displaying stage-specific influence on neurogenesis.

FIG. 1.

Notch involvement during ESC maintenance. (A) Notch inhibition attenuated nuclear localization of activated Notch (aNotch) in ESCs. The effect was more pronounced for −LIF than for +LIF, as seen after 1 day of inhibition. (B) ESC morphology for the +LIF condition was comparable between the dimethyl sulfoxide DMSO control and gSI treatment groups after 6 days in culture. With −LIF, however, cells exhibited an enlarged and flattened shape. (C) Under the +LIF condition, Oct4 expression (red) was maintained even after 6 days of sustained inhibition of Notch with gSI treatment and remained similar irrespective of the inhibitor concentration used. Although Oct4 expression diminished drastically under the −LIF condition, it was comparable irrespective of gSI treatment. (D) Notch inhibition for the +LIF condition did not show the presence of Numb in ESCs, while it increased the Numb level significantly under the −LIF condition, as seen after 1 day of inhibition. (E) Notch activation by either Jag1 or Dll4 (1 μg/ml each) induced differentiation in ESCs even under the +LIF condition. (F and G) qPCR showing Oct4 and Nanog expression under the +LIF or −LIF condition with ligand treatments. DAPI was used for the nuclear counterstain. Scale bar, 50 μm (A to E). Data are means ± SEM; n = 4 to 10. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 2.

Notch ligand bias and lineage commitment in ESCs. (A) qPCR showing an increase in Fgf5 expression with Jag1 (dose dependent) and decrease with Dll4 under the +LIF condition. For −LIF, only Dll4 displayed decreased expression compared to the control. (B) For +LIF, Jag1 treatment decreased both Bry (dose dependent) and Afp expression, and that of Nes was increased at 1 μg, while it was decreased at 10 μg of Jag1 compared to the control. Similarly, Dll4 decreased Afp (dose dependent) and Nes expression, while Bry remained unchanged. (C) For −LIF, Jag1 increased Nes and decreased Bry compared to the control, while Dll4 increased Bry at both doses and Afp at the low dose only. Data were presented as fold differences from the +LIF control, keeping its value at 1, and the statistical significance was calculated, comparing with the respective controls for both the +LIF and −LIF conditions. Data are means ± SEM; n = 4 to 10. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 3.

qPCR analyses of Notch receptor and downstream gene expression. (A) Notch receptor expression profile in ESCs under the +LIF and −LIF conditions. Statistical significance was calculated, comparing the individual receptor expression under both the +LIF and −LIF conditions. (B) Ligand treatments increased Hes1 expression (dose dependently) for +LIF and showed no influence for −LIF. Jag1 increased Hes5 expression under both the +LIF and −LIF conditions, while Dll4 decreased it. Conversely, Dll4 increased Hey1 expression, and Jag1 decreased it. Data were presented as fold differences from the +LIF control, keeping its value at 1, and the statistical significance was calculated in comparison with results for respective controls under both the +LIF and −LIF conditions. Data are means ± SEM; n = 4 to 10. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 4.

Notch participation during neural progenitor generation. (A and B) Flow cytometry quantification of cells on the 4th (A) (d4) or 7th (B) (d7) day of differentiation revealed an increased population of Sox1⁺ cells upon Jag1 treatment compared to the control level, while Notch inhibition did not influence it. (C) However, on day 2 (d2) of differentiation, flow cytometry quantification revealed Notch activation with Jag1 increasing the Sox1 population, while inhibition with gSI decreased the same, compared with results for respective controls. (D) Temporal influence of Notch activation and inhibition on generation of a neural progenitor (Sox1) population on day 4 of differentiation. Jag1 increased the Sox1⁺ population following its treatment on both the 0- to 2-day and 2- to 4-day time points, similar to results seen on day 2 of differentiation. However, gSI showed a contrasting response, inhibiting neural differentiation with treatment during the 0- to 2-day time interval and with no response in subsequent 2- to 4-day treatment. (E) However, on day 7, the Nes⁺ population was significantly higher with Notch inhibition than that with the DMSO vehicle control and Jag1. (F) Quantification of nes-EGFP cells on day 7 illustrated that Jag1 treatment increased the EGFP^low population while gSI treatment increased both the EGFP^high and total EGFP populations. Data are means ± SEM; n = 4 to 10. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 5.

Notch involvement during neural maturation. (A) Quantification of the MAP2⁺ population on day 14 of differentiation revealed gSI treatment enhancing neuronal differentiation at all the time points except the initial one (0 to 2 days). (B) However, the GFAP⁺ population under similar conditions was decreased at all the time points, indicating inhibition of gliogenesis. Numbers 1 to 6 represent different time intervals of 0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10, and 10 to 12 days of treatment, respectively. (C) Sustained inhibition of Notch (gSI) increased the TuJ1⁺ population on differentiation day 8, while activation with Jag1 decreased it. (D) Notch inhibition at all the time intervals except the initial one (0 to 2 days) also increased the MAP2⁺ population on day 8. (E) Notch inhibition augmented the number of neurites formed per individual neuron, along with enhanced neurite length and secondary arborization. (F) Images showing the structure of neurons monitored on day 16 following gSI treatment during days 0 to 8 and 8 to 16. The complete length of a neurite is shown by a dashed line. Arrowheads: white, starting point of a neurite from the cell body; orange, branch points in the neurite. Day has been abbreviated as “d.” Scale bar, 50 μm. Data are means ± SEM; n = 4 to 10. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 6.

Notch-mediated glial differentiation. (A) Quantification of MAP2⁺ and GFAP⁺ populations revealed the Jag1-CNTF combination was most suited for gliogenesis compared to other treatments, while neuronal differentiation was attenuated by the same. However, BMP2 diminished the overall neural differentiation, since both MAP2⁺ and GFAP⁺ populations were diminished with the treatment. (B) Immunofluorescence studies displaying MAP2⁺ and GFAP⁺ populations under different treatment conditions. Day is abbreviated as “d.” Scale bar, 50 μm. Data are means ± SEM; n = 4 to 10. ***, P ≤ 0.001.

FIG. 7.

Schematic representation of stage-specific Notch involvement (↓, activation; ⊥, inhibition) during lineage commitment and neurogenic progression from ESCs.