Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion

Abstract

Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or "proneural wave" accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation.

Keywords: EGF; Notch; lateral inhibition; proneural wave; reaction diffusion.

Fig. 1.

The molecular mechanisms of Notch-mediated lateral inhibition and proneural wave progression. (A) Notch signaling between two cells mediates the binary cell fate decision. (B) The differentiating cells prevent the neighboring cells from differentiating via Dl/Notch signaling. (C–F) The proneural wave sweeps across the NE sheet (blue). The NE cells express L’sc (green), triggering the differentiation from NE to NB (red). (C) The WT. (D and E) In clones mutant for EGF and Notch signaling (black lines), the proneural wave is eliminated and accelerated, respectively. (F) A schema showing the relative distributions of AS-C family (L’sc, Sc, and Ase), Rho and Dl expression, and the activities of EGF and Notch signaling. (G) A schema showing the relationship between the EGF signal (E), Notch signal (N), Dl expression (D), and AS-C expression (A). Black and red arrows are based on known observations and findings in this work, respectively.

Fig. 2.

Notch activity along the proneural wave. The proneural wave in vivo in late third instar larval brain (anterior to the left and dorsal to the top). (A) In cells expressing the active form of Notch (GFP-positive; green), L'sc (blue) is suppressed, and Dl (white) is induced (white arrow). (B) In pnt mutant cells expressing the active form of Notch, L'sc and Dl are suppressed (white arrow). Left shows the expression levels of Dl (red) and GFP (blue) along the arrows (mutant clones), and Right shows the expression levels of Dl (red) and GFP (blue) along the dashed arrows (background) in A and B. (C) L'sc (blue) and PntP1 (white) are reduced in da mutant clone visualized by the absence of GFP (green). (D) L'sc (blue) is eliminated and PntP1 (white) is reduced in clones homozygous for Df(1)260–1 visualized by the absence of GFP (green). (Scale bar: 50 μm.)

Fig. 3.

The four-component model. (A) The four-component model. (B–F) Results of in silico experiments visualizing A, N, and E (e_e = 0.2 and n_e = e_d = 1.0). Mutant clones are surrounded by white dotted lines. (B) The WT. (C) EGF mutant clone. (D) Notch mutant clone. (E) Dl mutant clone. (F) Dl EGF double-mutant clone. (D and E) Proneural wave is nonautonomously accelerated (arrowheads). (G–O) Results of in silico experiments in the absence of the feedbacks (e_e = n_e = e_d = 0). Mutant clones are surrounded by white dotted lines. (G–K) The results of the WT and mutant clones are very similar to those in B–F. (L and M) D is down-regulated in EGF mutant clone. (N and O) E is down-regulated in Notch mutant clone (arrow). (F and K) The residual A value inside the Dl EGF double-mutant clone is most likely caused by EGF diffused from the WT cells.

Fig. S1.

Expression of L'sc, PntP1, and Dl before proneural wave progression. Expression patterns of L'sc (blue), PntP1 (green), and Dl (white) before the initiation of the proneural wave at second instar. White dots indicate the boundary between the optic lobe and the central brain.

Fig. S2.

Behaviors of the four-component model at various e_a values: (A) e_a = 10, (B) e_a = 4, and (C) e_a = 1. Results of in silico experiments visualizing A in the WT and the presence of clones mutant for EGF, Notch, Dl, and EGF/Dl (e_e = 0.2 and n_e = e_d = 1.0). Phase diagrams showing speed of wave progression, acceleration of wave in Notch clone, and SDs of A and N by changing NP (d_t = d_c = 0.25 × NP) and EP (n_e = a_e = EP and e_e = 0.2 × EP).

Fig. S3.

Behaviors of the four-component model in the absence or presence of the feedbacks. Results of in silico experiments visualizing A in the WT and the presence of clones mutant for EGF, Notch, Dl, and EGF/Dl. (A) No EGF autoregulation (e_e = 0, n_e = 1.0, and e_d = 1.0). (B) No EGF regulation by Notch (e_e = 0.5, n_e = 0, and e_d = 1.0). (C) No Dl regulation by EGF (e_e = 0.2, n_e = 1.0, and e_d = 0).

Fig. S4.

Phase diagrams of the four-component model in the absence or presence of the feedbacks. Phase diagrams showing speed of wave progression, acceleration of wave in Notch clone, and SDs of A and N by changing NP (d_t = d_c = 0.25 × NP) and EP (n_e = a_e = EP and e_e = 0.2 × EP). (A) Standard condition (e_e = 0.2, n_e = 1.0, and e_d = 1.0). (B) No EGF autoregulation (e_e = 0, n_e = 1.0, and e_d = 1.0). (C) No EGF regulation by Notch (e_e = 0.5, n_e = 0, and e_d = 1.0). (D) No Dl regulation by EGF (e_e = 0.2, n_e = 1.0, and e_d = 0).

Fig. S5.

Behaviors of the four-component model in a hexagonal grid model. (A and B) The arrangement of cells are compared in (A) a square grid model and (B) a hexagonal grid model. In both cases, the distances between neighboring cells are 2 (dx = 2). (C) Results of in silico experiments visualizing A in the WT and the presence of clones mutant for EGF, Notch, Dl, and EGF/Dl (d_e = 0.67, d_t = 0.17, d_c = 0.25, and e_e = n_e = e_d = 0). (D–G) Phase diagrams showing (D) speed of wave progression, (E) acceleration of wave in Notch clone, and SDs of (F) A and (G) N by changing NP (d_t = 0.17 × NP and d_c = 0.25 × NP) and EP (a_e = EP). Orange dotted lines indicate the area in which NP and EP are equivalent, showing wave acceleration in N clone without showing salt and pepper patterns in A and N. The red and white rectangles indicate the conditions shown in H–L. (H–L) Salt and pepper and striped patterns of A and N at the wave front by changing the value of EP (NP = 1.8). (H) EP = 1.5, (I) EP = 1.3, (J) EP = 1.0, and (K and L) EP = 0.8. (H–K) The WT and (L) Notch mutant clone.

Fig. 4.

Salt and pepper pattern is canceled by EGF in silico and in vivo. (A) Notch activity as visualized by E(spl)mγ-GFP (green) and Dl expression (white) is found at the wave front expressing L'sc (blue). White brackets indicate the coexpression of GFP and Dl. Anterior is to the left, and dorsal is to the top. (B–D) Reducing (C) EGF diffusion (d_e = 0.85) or (D) EP (a_e = 0.85), A and N show fluctuations compared with the control (B). (B–D and I) Results of in silico experiments in the absence of the feedbacks (e_e = n_e = e_d = 0). (E–G) Maximum intensity projection images of E(spl)mγ-GFP (white) and L'sc (blue) in (E) control, (F) rho heterozygous, and (G) pnt heterozygous backgrounds. Ectopic GFP-positive cells are indicated by arrows. (H) Notch activity as visualized by NRE-dVenus exhibits a salt and pepper pattern (white; arrows). *Signals behind the proneural wave reflect the Notch activity in NBs. (I) A and N show fluctuations in clones in which EGF production is reduced (a_e = 0.5). Arrows indicate the cells in which A is up-regulated while N is down-regulated. Arrowheads indicate the cells in which A is down-regulated while N is up-regulated. (Scale bars: 50 μm in A and E–H.) (J–R) EGF signaling was partially reduced under the control of optix-Gal4 in the dorsal and ventral regions of the optic lobe as visualized by GFP (green in J, M, and P). (J–L) Control. (M–O) UAS-pnt RNAi^JF02227. (P–R) UAS-Ras^N17. (J, M, and P) L'sc expression became stochastic when EGF signaling was reduced as visualized by PntP1 (arrows and arrowheads in M and P). (K, N, and Q) Notch activity as visualized by E(spl)mγ-GFP (green) and L'sc expression (magenta) was compared at the dorsal regions of the optic lobe. (N and Q) L'sc and GFP showed stochastic expression when EGF signal was reduced (arrows and arrowheads, respectively). Note that the L'sc and GFP signals were largely complementary to each other. (L, O, and R) NB differentiation as visualized by Dpn expression (white) was not impaired in the absence of L'sc expression (magenta; arrows in O and R). (K, L, N, O, Q, and R) Maximum intensity projection images. (Scale bars: 50 μm in J, L, M, O, P, and R; 25 μm in K, N, and Q.)

Fig. S6.

Differential inactivation of EGF signaling by pnt RNAi. (A) EGF signaling was strongly reduced by optix-Gal4 UAS-pnt RNAi^JF02227 at 30 °C. GFP (green), L'sc (blue), and PntP1 (magenta). Expression of L'sc and PntP1 was completely lost, except for a small number of L'sc-positive cells in the lateral region of the optic lobe (arrow). (B–D) The effects of pnt RNAi on eye development were compared under the control of GMR-Gal4 at 25 °C. (B) Control. (C) UAS-pnt RNAi^JF02227 caused mild reduction in the eye size. (D) UAS-pnt RNAi^HMS01452 caused severe reduction in the eye size. (E) EGF signaling was strongly reduced by optix-Gal4 UAS-pnt RNAi^HMS01452 at 25 °C. GFP (green) and L'sc (blue). The proneural wave as visualized by L'sc expression was eliminated. Optic stalk is indicated by an arrow.

Fig. 5.

Phase diagrams of the four-component model. Results of in silico experiments in the absence of the feedbacks (e_e = n_e = e_d = 0). (A) Speed of wave progression, (B) acceleration of wave in Notch clone, and (C and D) SDs of A and N at the wave front, respectively, were examined by changing NP (d_t = d_c = 0.25 × NP) and EP (a_e = EP). Orange dotted lines indicate the area in which NP and EP are equivalent, showing wave acceleration in N clone without showing salt and pepper patterns in A and N. White and black dotted lines indicate the area in which NP is relatively larger or EP is relatively smaller showing salt and pepper patterns in A and N. The red rectangles indicate the conditions shown in E–G. (E–G) Salt and pepper and striped patterns of A and N at the wave front by changing the value of EP (NP = 1.8). (E) EP = 1.5, (F) EP = 1.1, and (G) EP = 0.8.

Fig. 6.

Wave acceleration mechanism in Notch mutant clone. (A–C) Results of in silico experiments in the absence of the feedbacks (e_e = n_e = e_d = 0). Notch mutant clones are indicated by white dotted lines. (A and B) E is activated when the proneural wave encounters Notch mutant cells (arrows in A) but quickly inactivated (arrow in B). (B) The peak values of E at the wave front and the widths of E activated areas (E > 0.09) are compared along the lines a and b. (C) The wave acceleration in Notch mutant clone is suppressed when a_d = 0. (D) PntP1 (white) is transiently up-regulated when proneural wave encounters Su(H) mutant cells (arrowheads; GFP-negative) but eventually down-regulated (arrows). The peak levels of PntP1 and the widths of PntP1-activated areas are compared along the lines a–c. The line d indicates the background level. (Scale bar: 50 μm.)

Fig. S7.

Wave acceleration in Dl mutant clone. (A) Wave acceleration in Dl mutant clone in silico (white dotted lines; e_e = n_e = e_d = 0). E is activated at the wave front within Dl mutant cells (arrows) but quickly inactivated. (B and C) In Dl mutant clones in vivo (arrows; GFP-negative), the proneural wave is accelerated as visualized by L’sc (blue). Additionally, EGF signaling as visualized by PntP1 (white) is transiently up-regulated but eventually down-regulated (arrows).

Fig. S8.

Notch forms a pulse wave. (A) The three-component model. (B–F) Results of in silico experiments visualizing A, N, and E. (B) The WT. (C–F) Mutant clones are surrounded by white dotted lines. (C) EGF mutant clone. (D–F) Notch mutant clones. E is activated when the proneural wave encounters Notch mutant cells (arrows in D) but quickly inactivated (arrow in E). Proneural wave is nonautonomously accelerated (arrowheads in E). (F) The wave acceleration is suppressed when ${\overline{a}}_{\mathit{n}}$ = 0. (G) The pulse waves of EGF and Notch activity positively and negatively controlling the wave progression, respectively. (H) A scheme showing that the reaction diffusion of EGF and Notch-mediated lateral inhibition promote the wave progression and salt and pepper pattern formation, respectively. In contrast, EGF and Notch negatively regulate the salt and pepper pattern formation and wave progression, respectively. When the signals of EGF and Notch are equivalent, the salt and pepper pattern is obscured.