A remarkably small number of signaling cascades are used across phyla to control cell specification. Thus, specificity must arise from combinatorial and cross-regulatory interactions among these pathways. Significant progress has been made in identifying the common immediate effectors for many of these conserved pathways, including those used during Ras- and Notch-dependent signaling. However, how these common pathways drive distinct cell fate choices in particular organs remains unclear.
Many of the common components within the Ras/mitogen-activated protein kinase (MAPK) and Notch/Delta signaling pathways have been identified and studied extensively in the developing Drosophila
compound eye [1
]. This is in large part because each of the 20 cells present in the approximately 750 repeating adult eye units (ommatidia) in this organ is stereotypically and sequentially recruited by the reiterative use of these two pathways. First, the R8 photoreceptor neuron is selected through Notch-dependent lateral inhibition, followed by stepwise recruitment of the R2/R5, R3/R4, R1/R6, and R7 photoreceptors (PRs), four cone cells (CCs), two primary pigment cells (PPCs), and approximately nine shared highly pigmented interommatidial cells by use of both Ras and Notch signaling [5
]. How Ras and Notch are differentially interpreted in these different cell types, however, is not understood.
Specification of the R7 photoreceptor provides a particularly useful model for exploring Ras and Notch signal integration in the fly eye. The R7 and CCs are the last cells to be recruited to ommatidial clusters before pupation, and classic studies have established that prior to their specification, these five cells comprise an 'R7 equivalence group' [10
]. All cells within the R7 equivalence group express Notch, the epidermal growth factor (EGF) receptor (EGFR), and Sevenless, a tyrosine kinase receptor that, like EGFR, signals via the Ras/MAPK pathway. The ligands Delta and EGF are made available from the previously specified PRs R1 and R6, but only one cell can directly contact Boss, the Sev ligand, present on the surface of the R8 PR [12
]. This cell, receiving higher Ras/MAPK levels, is driven towards an R7 fate, while the remaining cells that do not receive Sev signaling become epithelial CCs [2
]. These studies have led to the prevailing model that Ras/MAPK signaling is necessary and sufficient for neuronal versus non-neuronal cell fate decisions in this group of cells. However, more recent work in the fly eye has shown that Ras/MAPK levels also indirectly control Delta expression [18
], a neurogenic factor in much of the developing nervous system [20
], and mutants affecting Ras or Notch signaling cause only partial R7 versus CC fate switches [2
]. Thus, it is important to clarify the role of these signaling pathways in R7 versus CC fate choices, as it serves as a useful model for addressing how different levels of signaling mediate cell-specific fate decisions during development.
Two specific downstream targets of Ras and Notch within the R7 equivalence group are the transcription factors Prospero (Pros) and dPax2 (also known as sparkling
) or shaven
]. Pros is expressed in all five cells and is later up-regulated in the presumptive R7 by Sevenless [26
]. dPax2, on the other hand, is restricted to the four CC precursor cells, and is later expanded into the PPCs, the next cells to form in the eye [28
]. Enhancers partially reflecting these Pros and dPax2 expression patterns have been identified. Pros expression is directly up-regulated by the EGF/Ras/MAPK-activated ETS transcription factor PntP2, and is probably indirectly regulated by Notch signaling [26
]. dPax2, instead, is controlled by the combined direct inputs of PntP2 and the Notch/Su(H) activation complex [30
]. While these studies provide evidence that Pros and dPax2 are direct transcriptional targets of Ras and Notch signaling, the functional relevance of this regulation remains an unanswered question.
Here, we demonstrate that Pros and dPax2 are expressed at different levels in distinct subtypes of CC precursors, indicating that not all CCs are 'equivalent'. Further, we show that individual mutations in Pros or Pax2 cause minor changes in CC numbers, while their simultaneous removal prevents all CC recruitment, revealing that these factors combinatorially control CC fate determination. Mechanistically, we demonstrate that Pros maintains high-activated MAPK (pERK) levels in the R7 equivalence group, thus promoting neuronal PR fate within this cell population. Conversely, dPax2 transcriptionally suppresses Delta expression in CC precursors, serving as an anti-neuronal factor. Importantly, the integration of Pros and dPax2 functions via Ras and Notch signaling feedback is necessary to fully control neuronal versus non-neuronal cell fate decisions in the fly eye. Since Pros and dPax2 orthologs are co-expressed in many developmental systems requiring Ras/MAPK and Delta/Notch inputs, it is likely that these processes are evolutionarily conserved.