The Drosophila eye offers an excellent opportunity to understand how general developmental processes are subtly altered to result in specific cell fates. Numerous transcription factors have been characterized in the developing eye; most of these are active in overlapping subsets of cells. Mechanisms used to regulate transcription factors act at many levels, and include competition for cognate binding sites, post translational modification, transcriptional regulation and cofactor availability. In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prosperoenhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable Yan (ACT) cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. We sequenced the eye-specific enhancer of lozenge in three Drosophila species spanning 17 million years of evolution and found complete conservation of three ETS consensus binding sites. We show that lozengeexpression increases as cells differentiate, and that Yan (ACT) blocks this upregulation at the level of transcription. We find that expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of Yan (ACT). These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. We propose that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development.

Physical and functional characteristics of the RUNX family of transcription factors are conserved between vertebrates and the Drosophila protein Lozenge. The runt-homology domain responsible for DNA binding and also the C-terminus are both nearly identical between the two proteins. The mammalian and fly proteins heterodimerize with a non-DNA binding partner protein to form a core binding factor essential for gene regulation during cell differentiation. The mammalian protein RUNX1 (AML1/PEBP2alphaB) interacts with the transcription factor Ets-1 to increase DNA binding and transactivation potential. Alternative splicing of the mammalian RUNX1 removes a domain required for this cooperative transactivation. In this work we determine the structure of the lozenge transcription unit and map 21 mutations. We show that the lozenge transcript is alternatively spliced during eye development to remove an Ets interaction domain. Emphasis is placed on Pointed the Drosophila homolog of the vertebrate Ets-1 protein; both Lozenge and Pointed proteins are needed for the activation of prospero expression. We use site-directed mutagenesis and yeast two-hybrid analysis to show that conserved amino acids within the alternate Lozenge exon are important for interaction with Pointed. Furthermore, the ectopic expression of Lozenge is sufficient to rescue Prospero expression in the presence of the Pointed competitor, Yan(ACT). We show that both lozenge isoforms are expressed during eye development and that the relative ratio of the transcripts for the two isoforms is sensitive to changes in Ras activity. We suggest that during eye development, Lozenge isoforms function in divergent roles, either interacting with Pointed on downstream targets or by functioning independently to establish distinct cell fates.

No abstract

Mutations in the lozenge gene of Drosophila melanogaster elicit a pleiotropic set of adult phenotypes, including severe compound eye perturbations resulting from the defective recruitment of photoreceptors R1/6 and R7, cone and pigment cells. In this study, we show that excessive patterned apoptosis is evident at the same developmental stage in these lozenge mutants. In lozenge null mutants, apoptosis occurs prior to lozenge-dependent cell fate specification. A second gene, D-Pax2, genetically interacts with lozenge. Interestingly, D-Pax2 mutants also exhibit increased cell death, but slightly later in development than that in lozenge mutants. Although expression of the caspase inhibitor p35 eliminates death in both lozenge and D-Pax2 mutants, the lozenge mutant eye phenotypes persist because other normal Lozenge functions are still lacking. D-Pax2 eye phenotypes, in contrast, are dramatically altered in a p35 background, because cells that normally differentiate as cone and primary pigment cells are subsequently transformed into second-ary pigment cells. This study leads us to propose that Lozenge, aside from its known role in gene regulation of cell-specific transcription factors, is required to contribute to the repression of cell death mechanisms, creating a permissive environment for the survival of undifferentiated cells in early eye development. Lack of lozenge expression increases the likelihood that an undifferentiated cell will initiate its default death program and die prematurely. The ectopic cell death evident in D-Pax2 mutants appears to arise from the cell fate transformation of cone cells into secondary pigment cells, either autonomously or as a result of defective signalling.

Authentic assessment exercises are similar to real-world tasks that would be expected by a professional. An authentic assessment in combination with an inquiry-based learning activity enhances students' learning and rehearses them for their future roles, whether as scientists or as informed citizens. Over a period of 2 years, we experimented with two inquiry-based projects; one had traditional scientific inquiry characteristics, and the other used popular culture as the medium of inquiry. We found that activities that incorporated group learning motivated students and sharpened their abilities to apply and communicate their knowledge of science. We also discovered that incorporating popular culture provided ““Millennial”” students with a refreshing view of science learning and increased their appetites to explore and elaborate on science.