Vertebrate and invertebrate neural progenitors are temporally patterned to generate a great diversity of neural types in a birth-order dependent manner. Series of temporal transcription factors (TTF) were found to be sequentially expressed in Drosophila neuroblasts, and they are proposed to form transcriptional cascades to control the sequential generation of different neural types. However, whether the cross-regulations inferred from mutant phenotypes are direct transcriptional regulations haven't been demonstrated. Furthermore, the cross-regulations among TTFs are often not sufficient for the temporal transitions, suggesting other mechanisms are at play to regulate the temporal progression. We use the Drosophila medulla neuroblasts to study these questions. In the previous R01 period, we identified molecular mechanisms controlling transitions to the Slp and Ey temporal stages, and also identified a comprehensive list of novel temporal transcription factors through single-cell RNA sequencing. In this renewal application, we present our preliminary data of single-nuclear ATAC seq, which revealed the dynamic chromatin accessibility during temporal patterning of medulla neuroblasts. Through analyzing the differentially accessible regions, we identified the possible enhancers controlling the temporal expression patterns of TTF genes. Through integration of scRNA-seq and snATACseq and a combination of reporter assays, genetic analysis and Dam-ID experiments, we propose to elucidate the transcriptional regulatory networks controlling the sequential temporal transitions in great detail. Furthermore, we found that different epigenetic factors are required at different steps of temporal patterning. We propose to further examine how they regulate the dynamic chromatin accessibility and how they are recruited to specific target genes during temporal patterning. Finally, we propose to examine the fundamental molecular mechanisms that coordinate the growth/proliferation with TTF cascade progression, and will test our hypothesis that early TTFs have different roles in controlling growth/proliferation than late TTFs through a combination of approaches.