Eukaryotic cellular tissues are generally comprised of several cell types, many of which may be derived from a common pool of precursor cells. How such developmentally equivalent cells become distinct from one another remains a fundamental question in developmental biology. Specification of cell fates involves the interpretation of multiple signalling pathways by individual cells. The developing eye of Drosophila has been extensively used as a model system to determine how common signalling pathways can induce the generation of cellular diversity. In particular, specification of the R7 photoreceptor cell fate has been a principal paradigm for elucidation of how cell fates are established in response to signalling cues .
The adult Drosophila eye is comprised of approximately 800 ommatidia, with each ommatidium containing eight photoreceptor neurons surrounded by a collection of non-neuronal support cells . The eye begins its development from the eye imaginal disc epithelium during mid-third larval instar, with the morphogenetic furrow progressing anteriorly across the disc, marking the onset of cell differentiation and pattern formation . Photoreceptor R8 is the first cell established in the eye, its recruitment mediated by signalling events coordinated by the furrow [3, 4]. Three pairs of photoreceptors, R2/5, R3/4 and R1/6, are then subsequently recruited to each ommatidial cluster, their recruitment being dependent upon reiterative induction of the epidermal growth factor receptor (EGFR) signalling pathway [5–8]. The last photoreceptor to be recruited is the R7 cell. Addition of non-neuronal lens secreting cone cells, supporting pigment cells, and the generation of sensory bristle cells make up the full complement of ommatidial cells [4, 9].
Induction of the R7 cell has been the most extensively studied cell differentiation event in the eye. With respect to common signalling events, the Notch (N) signalling pathway and the receptor tyrosine kinases (RTKs), EGFR and Sevenless (Sev), have been shown to be necessary for induction of the R7 fate [10–15]. Loss of N signalling has been shown to cause the R7 precursor cell to adopt an R1/R6 cell fate. Conversely, ectopic N activation in R1/6 cells is sufficient to covert these cells into R7 cells [11, 15]. The ability of N to potentiate R7 development is dependent on the expression of the N ligand, Delta, in R1/6 photoreceptors [11, 15]. Moreover, N may induce R7 fate differentiation in the presumptive R7 cell by both activating R7-cell-specific determinants, and repressing R8 cell determinants [11, 14, 15]. Successive episodes of EGFR activation of the Ras/MAPK (Mitogen-Activated Protein Kinase) signalling cascade has been shown to be a requirement for recruitment of all photoreceptor neurons to the ommatidium, including the R7 cell [5, 6, 8]. In contrast, Sev signalling is restricted to the presumptive R7 cell, with loss of Sev signalling specifically resulting in the trans-determination of the presumptive R7 cell into a non-neuronal cone cell [10, 12, 13]. While Sev and EGFR both feed into the same signal transduction pathway, high levels of RTK activation in the presumptive R7 cell may be required to overcome repressive mechanisms specific to the R7 cell itself (reviewed in ). In the presumptive R7 cell, high levels of RTK signalling result in the expression of a novel nuclear gene phyllopod (phyl) [16, 17]. Phyl functions as an adaptor protein in the R7 nucleus, recruiting the neuronal inhibitor Tramtrack (Ttk) into a complex with Seven in absentia (Sina) and Ebi [18–21]. Ttk RNA is alternatively spliced, giving rise to two zinc-finger DNA binding proteins, Ttk69 and Ttk88. Both Ttk isoforms share a common N-terminal region containing a BTB/POZ (Broad Complex Tramtrack Bric-a-Brac/Pox virus and Zinc finger) domain, but have alternative sets of Cys2-His2 zinc-fingers in the carboxyl fragment, resulting in the two isoforms having different DNA-binding specificities . Both Ttk isoforms function to block neuronal fate specification in the third instar developing eye disc, and are presumed to be antagonists of RTK signalling, since overexpression of either isoform results in a failure of photoreceptor recruitment [21–24]. The recruitment of Ttk88 into the Sina-Ebi complex in R7 precursor cells leads to Ttk88 ubiquitination and post-translational degradation by proteolysis . Targeted degradation of Ttk88 relieves the inhibition of the neuronal cell fate, allowing R7 fate specification to proceed. Evidence suggests that Ttk69 is also targeted for degradation through induction of RTK signalling [19, 25, 26]. Additional studies have also implicated the RNA-binding protein Musashi (Msi) in the translational inhibition of Ttk69 in R1/6/7 precursor cells in the developing eye [27, 28]. While Ttk needs to be degraded for R7 fate specification to proceed, its presence is required for the formation of non-neuronal cone and pigment cells, highlighting the importance for tight regulatory control of cell-specific transcription factors in order to correctly specify cell fate within the developing eye disc [19, 26, 29].
The R7 cell arises from a population of undifferentiated cells surrounding the already recruited five-cell neuronal pre-cluster of R8/2/5/3/4 cells. These unspecified cells have undergone a second round of mitosis, an event required for re-population of the epithelium for the additional recruitment of the remaining photoreceptors, and non-neuronal supporting cells. The recruitment and differentiation of cells arising from the second mitotic wave requires the combinatorial inputs of N, EGFR and Sev signalling [30–33]. Additionally, these cells require the expression of Lozenge (Lz), a member of the RUNX family of transcription factors . RUNX proteins are critical in development, since loss of RUNX protein function can lead to stomach cancer , the development of acute myeloid leukemia  or other severe developmental defects .
In its role in eye development, Lz has been described as a pre-patterning factor, since its expression within a pool of equipotent undifferentiated cells is required for the subsequent recruitment and differentiation of R1/6 and R7 cells, cone and pigment cells [30, 38]. In the absence of Lz function, these cell types fail to correctly differentiate and excessive apoptosis in third-instar eye discs can be observed [38–42]. Lz, in combination with other factors, is required to regulate a number of cell specific transcription factors expressed posterior to the second mitotic wave. For instance, Lz acts in a combinatorial manner with Yan, PointedP2 and Suppressor of Hairless (Su(H)) to restrict D-Pax2/shaven expression to the cone cell precursors in third instar eye discs [30, 43]. The regulation of prospero (pros) expression in R7 and cone cells is also dependent upon a combination of upstream transcription factors, including Lz, Pointed and Yan [32, 41]. Lz has also been shown to negatively regulate seven-up in R7 and cone cells [34, 40], deadpan (dpn) expression in cone cells , and Bar expression in R1 and R6 cells [34, 40].
Although studies have demonstrated the importance of Lz in the specification of differentiated cell types in the eye disc after the second mitotic wave, lz gene regulation itself is not fully understood. While lz expression is initially activated in undifferentiated cells by Sine Oculis (So) and Glass (Gl) , evidence suggests that lz expression levels are up-regulated in differentiating cell types [39, 41]. There is also some evidence to suggest that the Ras1/MAPK signalling pathway blocks the up-regulation of lz expression in undifferentiated cells through repression by the Yan protein [39, 41].
Additional complexity to the regulation of lz gene expression is added by the findings that lz mRNA is alternatively spliced during eye development, producing a full length isoform (826aa; c3.5) and an isoform lacking exon V (705aa; Δ5 ). Exon V encodes a conserved ETS interaction domain, and yeast two hybrid screens showed the direct interaction between Lz and the ETS factor PointedP2, with this interaction removed upon site directed mutagenesis of ETS interacting sequences within this exon . Exon V is critical for the development of presumptive R7 cells in the third instar eye disc, since R7 precursor cells can still develop in a severely truncated lz mutant with this exon intact.
In this study, we show that R7 development in third instar eye discs is dependent upon Lz function, and that Ttk69 may play a role in the direct or indirect repression of lz gene expression in cell types competent to develop as R7 cells. We show that loss of Ttk69 function results in the development of ectopic R7 cells in third instar eye development. These ectopic R7 cells are dependent upon Lz function for their development. Conversely, overexpression of Ttk69 results in loss of all lz-dependant differentiated cell types in the developing eye disc along with a marked decrease in Lz expression in both undifferentiated and differentiated cell types. Interestingly, by re-introducing Lz into a developing eye where Ttk69 is over-expressed, we could partially rescue cells expressing the lz-dependent factors Bar, Pros and Cut. We could also partially rescue the expression of the R7-cell-specific marker Klingon. Together, these results suggest that the loss of cells in the Ttk69 over-expressing lines was largely due to the removal of Lz function.
Additionally, we show that Sina and Msi, factors upstream of Ttk69 in eye development, play a Ttk-dependent role in lz gene regulation in R7 precursor cells. The elucidation of the function of Ttk69 in lz gene repression in a subset of cells provides a possible mechanism for the tight control of lz expression required for the correct differentiation of cell types during early eye development.