Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation

Abstract

Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, we show that in response to planar cell polarity (PCP) signals, Ral modulates asymmetric Notch signaling in the Drosophila eye. Specification of the initially equivalent R3/R4 photoreceptor precursor cells in each developing ommatidium occurs in response to a gradient of Frizzled (Fz) signaling. The cell with the most Fz signal (R3) activates the Notch receptor in the adjacent cell (R4) via the ligand Delta, resulting in R3/R4 cell determination and their asymmetric positions within the ommatidium. Two mechanisms have been proposed for ensuring that the cell with the most Fz activation sends the Delta signal: Fz-dependent transcriptional upregulation in R3 of genes that promote Delta signaling, and direct blockage of Notch receptor activation in R3 by localization of an activated Fz/Disheveled protein complex to the side of the plasma membrane adjacent to R4. Here, we discover a distinct mechanism for biasing the direction of Notch signaling that depends on Ral. Using genetic experiments in vivo, we show that, in direct response to Fz signaling, Ral transcription is upregulated in R3, and Ral represses ligand-independent activation of Notch in R3. Thus, prevention of ligand-independent Notch activation is not simply a constitutive process, but is a target for regulation by Ral during cell fate specification and pattern formation.

The ADAM family

“…This form of ligand-independent Notch cleavage has been observed in fly eye disks where in developing ommatidia, R3 precursor cells express the Notch ligand Delta and signal to the adjacent R4 precursors to become R4 cells. 2 Cho and Fischer recently determined that Ral GTPase prevents ligand-independent activation of Notch in pre-R3 cells, 18 demonstrating the importance of regulating the different types of Notch cleavage. The first ADAM 10 homolog to be characterized in the context of Notch cleavage was the product of the Drosophila gene kuzbanian (kuz).…”

Ral inhibits ligand-independent Notch signaling in Drosophila

“…Nevertheless, we determined that loss of Ral activity results in Notch receptor activation independent of ligand binding. 19 The main evidence for this lies in four observations. First, the specific defects in Ral mutant eyes were typical …”

“…30,32 We found that Ral defines a distinct pathway by which R3 is biased to become the Delta signaling cell. 19 In response to Frizzled activation, elevated Ral activity in R3 prevents ligand-independent Notch activation in pre-R3, thereby helping to tip the scales in favor of pre-R3 becoming the signaling cell. The main evidence for this idea is 2-fold.…”

“…26,27 We determined that we identified Ral mutants in our screen because Ral is a negative regulator of Notch signaling. 19 We do not understand the mechanism by which Epsin overexpression disrupts eye development and so we cannot say precisely why loss of Ral activity enhances the eye defects. Nevertheless, we determined that loss of Ral activity results in Notch receptor activation independent of ligand binding.…”

“…Ral defines a unique pathway for insuring that pre-R3 becomes the Delta signaling cell. 19 Ral transcription also depends on Fz, and Ral activity prevents activation of Notch receptor that is not bound to ligand. 19 Loss of Ral activity (bottom) results in symmetric R3/R4 pairs and ligand-independent Notch activation.…”