Dispatch R683

Dispatches

Notch Signalling: Receptor cis-Inhibition To Achieve Directionality Lateral inhibition, by which single cells become distinct from their neighbours, can be mediated by Notch signalling during animal development. Signalling directionality is presumably achieved by downregulation of the Notch ligand in signal-receiving cells. New evidence suggests that cis-inhibition of the receptor in the ligand-sending cell might also provide directionality. David del A´lamo and Franc¸ois Schweisguth*

recruitment signal and the final number of cells recruited is controlled by Notch lateral inhibition (Figure 1A). Dl is expressed in the newly-recruited R-cells and signals to the surrounding undifferentiated progenitor cells to inhibit them from adopting the R-cell fate [5]. Besides this, two other directional Notch signalling events take place during R-cell specification: R3 signals to R4 and R1/R6 signal to R7 [5]. The

Morphogenetic furrow

A Anterior

Posterior

Ommatidial development EGFR

DI

DI

Dl

B R1/R6/R7 specification R1

N

cis

cis

?

?

Dl

R2

R3

R5

R4

R8

R8

tra

Dl

R6

N

R1 R7 R6

C trans-inhibition model R6

R1

N

Dl

Dl

N

D cis-inhibition model R1

N

R7

R6

Dl

Dl

N

N

Dl

Dl

Dl

R7

R7

R7

?N

ns

Notch signalling is used reiteratively for a large variety of processes during animal development. One conserved principal function of Notch signalling is the ability to distinguish one cell from a group of equivalent ones in a process referred to as ‘lateral inhibition’ [1]. Thereby, the ‘chosen cell’ sends a signal, the Notch ligand Delta (Dl), to the neighbouring equivalent cell(s) and precludes them from adopting the same fate by activating the Notch receptor in these cells. Although the establishment of the directionality of the signal is complex and not completely understood, it is commonly accepted that a key mechanism in this process is a negative feedback loop in which cells receiving the signal down-regulate Dl expression, thus reducing their ability to signal back [2]. This mechanism is called ‘trans-inhibition’ as Dl in the signal-sending cell inhibits Dl in the signal-receiving cell [1]. New evidence presented by Miller and colleagues [3] in this issue of Current Biology challenges this view and demonstrates that ‘cis-inhibition’ of the Notch pathway by Dl in the signal-sending cell also plays a role — at least during Drosophila eye development. The Drosophila compound eye is formed by about 800 units called ‘ommatidia’, each of which comprises 8 photoreceptor neurons (labelled R1 to R8) and several accessory cells [4]. During eye development, after the ommatidium founder cell R8 has been specified, the rest of the photoreceptors (R1–R7) are recruited by the reiterative use of the epidermal growth factor (EGF) receptor and Notch pathways. EGF is the

latter constitutes a rather atypical example of Notch directional signalling in the sense that two ‘chosen cells’, R1 and R6, signal redundantly to the third one, R7, in order to inhibit the R1/R6 fate in this cell [6,7]. Miller and colleagues [3] cleverly exploit this peculiarity to put the trans-inhibition model to the test. R1, R6 and R7 are the last photoreceptors to be recruited into the ommatidial cluster. R1 and R6 are recruited together and start expressing Dl. When the last photoreceptor, R7, is recruited, Dl coming from R1 and R6 activates the R7 fate (or inhibits the R1/R6 fate). These three cells form

N

Current Biology

Figure 1. Notch signalling during Drosophila eye development. (A) Photoreceptor (R-cell) recruitment starts with specification of R8 by Notch-mediated lateral inhibition in the morphogenetic furrow. Successive rounds of EGF receptor signalling (recruitment signal) and Notch activation (inhibitory signal) lead to sequential recruitment of all other R-cells. R1/R6 and R7 are the last photoreceptors to be recruited. (B) Signalling by the Notch ligand Dl from R1/R6 activates Notch in R7. Reversal of the signal direction can, in principle, be avoided by two mechanisms: trans-inhibition of Dl in the signal-receiving cell or cis-inhibition of Notch in the signal-sending cell. (C) According to the trans-inhibition model, removal of Dl from only one cell of the R1/R6 pair has no effect on cell fate as Dl provided from the other cell of the pair is able to activate Notch which, in turn, down-regulates Dl. (D) The cis-inhibition model predicts that removal of Dl from only one cell of the pair has no effect on R7 fate, as it can be provided by the remaining wild-type cell. Nevertheless, as Dl is no longer cis-inhibiting Notch in the Dl mutant cell, Dl coming from R7 is now able to signal and transform this mutant cell into R7. This is indeed what Miller and colleagues [3] have observed.

Current Biology Vol 19 No 16 R684

an equivalence group as any cell with active Notch signalling will adopt the R7 fate while any cell with inactive Notch will adopt the R1/R6 fate (Figure 1B). Although R7 also expresses Dl once recruited, the signal always goes from R1/R6 to R7 [6,7]. A classic view of the problem assumes that directionality of the signal from R1/R6 to R7 relies on the early expression of Dl in R1/R6, which in turn down-regulates Dl expression in R7. If this view is correct, removing Dl from only one cell of the R1/R6 pair would cause no fate changes at all, as a Dl signal could still be provided by the remaining wild-type cell (Figure 1C). However, this experiment, performed by Miller and colleagues [3], shows a very interesting result: removing Dl from only one of the cells of the R1/R6 pair has no effect on the fate of R7, as predicted by the classical model, but it unexpectedly causes the mutant cell to become R7 as well. This fate transformation is dependent on Dl activity in the endogenous R7. Consistently, when R7 and either R1 or R6 are mutant for Dl, no fate transformation is observed. The other cell of the R1/R6 pair, which is wild type for Dl, should also be exposed to the signal from R7, but it does not respond. This result is in agreement with a model in which Dl cis-inhibits Notch in R1/R6 and precludes a reversal of the signal (Figure 1D). Why, then, is Notch not cis-inhibited by Dl in R7? By the time R7 starts expressing Dl, Notch has already received the signal from R1 and R6 and the R7 fate is already established. Indeed, forced early expression of Dl in the R7 precursor causes this cell to adopt the R1/R6 fate [3]. It is well established both in vertebrates and Drosophila that overexpression of the ligands leads to inhibition of Notch signalling in the overexpressing cells [8,9]. However, a physiological role for cis-inhibition in attenuating Notch signalling has only been described during wing margin development, a process in which the involved cells both send and receive the signal and no unique directionality is specified [9]. This is due, at least in part, to the fact that cis-inhibition is a difficult mechanism to address experimentally. During lateral inhibition, experimental removal of Dl from signal-sending cells leads to Notch activation and cell fate

transformation that could be explained either by lack of inhibition of Notch in the same cell (cis-inhibition) or lack of inhibition of Dl in the surrounding cells (trans-inhibition). It is thus of great importance — as Miller and colleagues have done [3] — to choose a system in which two equivalent and redundant signal-sending cells can be compared side by side in order to distinguish between these two possibilities. If the role of cis-inhibition has remained somewhat obscure, the molecular mechanism by which ligands are able to inhibit Notch is not more straightforward. The simplest explanation involves direct physical interaction and, indeed, interaction between Notch and its ligands has been observed both inter- and intra-cellularly [8,10]. Furthermore, recent results suggest that the same domains in Notch and the ligands are responsible for trans-activation and cis-inhibition and two modes of molecular interaction (parallel and antiparallel) could result in inhibition versus activation [11]. In any case, it is not clear what the effect of intracellular interaction of Notch and its ligands is. Cell culture assays have shown that Notch can be trapped inside the cell, making it impossible for it to receive signals from surrounding cells [8], although studies in Drosophila suggest that cis-inhibitory interactions could take place at the plasma membrane [10]. Whether cis-inhibition has a prominent role in other events controlled by Notch directional signalling or not still remains to be addressed. Strikingly, Miller and colleagues [3] show that removal of Neuralized (Neur), an E3-ubiquitin ligase required for Dl signalling [12,13], indeed disrupts the ability of Dl to signal and, more interestingly, R1/R6 cells mutant for neur do not acquire R7 fate. This suggests that Neur is not involved in the process of Notch cis-inhibition by Dl. This interpretation is consistent with described results obtained with a modified form of Serrate, the other ligand of Notch in Drosophila. This modified version of Serrate carries a deletion that renders it incapable of interaction with Neur and, when overexpressed, it is unable to trigger Notch trans-activation, as expected, but it retains its cis-inhibitory activity,

suggesting again that Neur may not be required for this aspect of ligand function [10]. If Neur proves to be necessary only for Dl trans-activation activity, Miller and colleagues [3] might have uncovered an excellent tool to study the phenomenon of Notch cis-inhibition in other contexts. References 1. Bray, S.J. (2006). Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678–689. 2. Heitzler, P., and Simpson, P. (1991). The choice of cell fate in the epidermis of Drosophila. Cell 64, 1083–1092. 3. Miller, A.C., Lyons, E.L., and Herman, T.G. (2009). Cis-inhibition of Notch by endogenous Delta biases the outcome of lateral inhibition. Curr. Biol. 19, 1378–1383. 4. Wolff, T., and Ready, D.F. (1993). Pattern formation in the Drosophila retina. In The Development of Drosophila melanogaster, M. Bate and A. Martinez-Arias, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Press), pp. 1277–1326. 5. Doroquez, D.B., and Rebay, I. (2006). Signal integration during development: mechanisms of EGFR and Notch pathway function and cross-talk. Crit. Rev. Biochem. Mol. Biol. 41, 339–385. 6. Tomlinson, A., and Struhl, G. (2001). Delta/Notch and Boss/Sevenless signals act combinatorially to specify the Drosophila R7 photoreceptor. Mol. Cell 7, 487–495. 7. Cooper, M.T., and Bray, S.J. (2000). R7 photoreceptor specification requires Notch activity. Curr. Biol. 10, 1507–1510. 8. Sakamoto, K., Ohara, O., Takagi, M., Takeda, S., and Katsube, K. (2002). Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev. Biol. 241, 313–326. 9. Micchelli, C.A., Rulifson, E.J., and Blair, S.S. (1997). The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124, 1485–1495. 10. Glittenberg, M., Pitsouli, C., Garvey, C., Delidakis, C., and Bray, S. (2006). Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis. EMBO J. 25, 4697–4706. 11. Cordle, J., Johnson, S., Tay, J.Z., Roversi, P., Wilkin, M.B., de Madrid, B.H., Shimizu, H., Jensen, S., Whiteman, P., Jin, B., et al. (2008). A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition. Nat. Struct. Mol. Biol. 15, 849–857. 12. Lai, E.C., Deblandre, G.A., Kintner, C., and Rubin, G.M. (2001). Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta. Dev. Cell 1, 783–794. 13. Pavlopoulos, E., Pitsouli, C., Klueg, K.M., Muskavitch, M.A., Moschonas, N.K., and Delidakis, C. (2001). neuralized encodes a peripheral membrane protein involved in delta signaling and endocytosis. Dev. Cell 1, 807–816.

Institut Pasteur, CNRS URA2578, 25 rue du Dr. Roux, 75724 Paris CEDEX 15, France. *E-mail: [email protected]

DOI: 10.1016/j.cub.2009.07.025