A context-dependent bifurcation in the Pointed transcriptional effector network contributes specificity and robustness to retinal cell fate acquisition.

Spatiotemporally precise and robust cell fate transitions, which depend on specific signaling cues, are fundamental to the development of appropriately patterned tissues. The fidelity and precision with which photoreceptor fates are recruited in the Drosophila eye exemplifies these principles. The fly eye consists of a highly ordered array of similar to 750 ommatidia, each of which contains eight distinct photoreceptors, R1-R8, specified sequentially in a precise spatial pattern. Recruitment of R1-R7 fates requires reiterative receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling mediated by the transcriptional effector Pointed (Pnt). However the overall signaling levels experienced by R2-R5 cells are distinct from those experienced by R1, R6 and R7. A relay mechanism between two Pnt isoforms initiated by MAPK activation directs the universal transcriptional response. Here we ask how the generic Pnt response is tailored to these two rounds of photoreceptor fate transitions. We find that during R2-R5 specification PntP2 is coexpressed with a closely related but previously uncharacterized isoform, PntP3. Using CRISPR/Cas9-generated isoform specific null alleles we show that under otherwise wild type conditions, R2-R5 fate specification is robust to loss of either PntP2 or PntP3, and that the two activate pntP1 redundantly; however under conditions of reduced MAPK activity, both are required. Mechanistically, our data suggest that intrinsic activity differences between PntP2 and PntP3, combined with positive and unexpected negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of compromised RTK signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify R1, R6 and R7 fates, the Pnt network resets to a simpler topology in which PntP2 uniquely activates pntP1 and auto-activates its own transcription. We propose that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments. Author summary Properly patterned tissues require cells transit from a multipotent state to diverse differentiated states in a precise spatiotemporal manner. Meanwhile the programs that direct cell fate adoption must be reliable despite genetic and nongenetic variation. In this study we use the Drosophila photoreceptors as a model system for understanding how specific and robust cell fate transitions are achieved. The specification of seven distinct photoreceptors R1-R7 requires repetitive inductive signaling from the receptor tyrosine kinase (RTK)/ mitogen-activated protein kinase (MAPK). The transcription factor Pointed (Pnt) operates downstream of MAPK to initiate the changes in gene expression appropriate to the particular transition. We asked how the generic MAPK/Pnt signal contributes to different photoreceptor fates. Previous work showed that R1-R7 photoreceptor specification can be subdivided into two rounds that experience different RTK signaling strengths. We find distinct Pnt regulatory networks operate in the two rounds, with the first round network incorporating a novel unstudied Pnt isoform, PntP3. Its inclusion stabilizes developmental transitions when signaling is reduced. We compare and contrast the expression patterns and transactivation potentials of the Pnt isoforms and uncover a web of transcriptional cross-regulation between them. Based on these explorations, we propose that the use of distinct Pnt network topologies provides an adaptive mechanism that permits reliable cell fate transitions under different MAPK signaling environments.