Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila

Reviewer #1 (Public Review):

In this manuscript, the authors are building on their previous work showing Delta-Notch regulates the entrance and exit from embryo-larval quiescence of neural stem cells of the central brain (called CB neuroblasts (NB) (PMID: 35112131)). Here they show that continuous depletion of Notch in NBs from early embryogenesis leads to cycling NBs in the adult. This - cycling NBs in the adult - is not seen in controls. The assumption here is that these Notch-RNAi NBs in adults are those that did not undergo terminal differentiation in pupal development. The authors show that Notch is activated by its ligand Delta which is expressed on the GMC daughter cell and on cortex glia. They determine that the temporal requirement for Notch activity is 0-72 hours after larval hatching (ALH) (i.e., 1st instar through mid-3rd instar at 25C). In NBs/GMCs depleted for Notch, early temporal markers were still expressed at time points when they should be off and late markers were delayed in expression. These effects were observed in ~20-40% of NBs (Figures 5 and 6). Through mining existing data sets, they found that the early temporal factor Imp - an RNA binding protein - can bind Delta mRNA. They state that Delta transcripts decrease over time (without any reference to a Figure or to published work), leading to the hypothesis that Delta mRNA is repressed by the late temporal factors. Over-expressing late factors Syp or E93 earlier in development leads to downregulation of a Delta::GFP protein trap. These results lead to a model in which Notch regulates expression of early temporal factors and early temporal factors regulate Notch activity through translation of Delta mRNA.

There are several strengths of this study. The authors report rigorous measurements and statistical analyses throughout the study. Their conclusions are appropriate for the results. Data mining revealed an important mechanism - that Imp binds Delta mRNA - supporting the model that early temporal factors promote Delta expression, which in turn promotes Notch signaling.

There are also several weaknesses:
1. The activation of Notch in NBs by Delta in GMCs was already shown by this group in their Dev 2022 paper, reducing some of the impact of this study.
2. The authors do not explain their current results in context of their prior paper (2022 Dev) until the Discussion, but this would be useful to read in the Introduction. Similarly, it would be good to mention that in the 2022 paper, they find a significant number of wor>Notch RNAi NBs at 2 AHL that are cycling. Are the adult Notch RNAi in this study descended from those NBs at 2 hours ALH in the 2022 study? In other words, how does the early requirement for Notch between 0-72 hours ALH reported in the current study relate to the Notch-depleted NBs identified in the 2022 paper?
3. Most of the experiments rely upon continuous depletion of Notch from embryonic stage 8 until adulthood using the wor-GAL4 driver. There is no lineage tracing of this driver and there is no citation about the published expression pattern of this driver. The inclusion of these details is important for a broad audience journal.
4. Most of the experiments utilize a single RNAi transgene for Notch, Delta, Imp, Syp, E93. There are no experiments demonstrating the efficacy of the RNAi lines and no references to prior use and/or efficacy of these lines.

An appraisal: The authors use temperature shifts with Gal80TS to show that Notch is required between 0-72 hours ALH. They show with the use of known markers of the temporal factors and Delta protein trap, that Imp promotes Delta protein expression and the later temporal factors reduce Delta, although the molecular mechanisms are not clearly delineated. Overall, these data support their model that the reduction of Delta expression during larval development leads to a loss of Notch activity.

As noted in the Discussion, this study raises many questions about what Notch does in larval CB NBs. For example, does it inhibit Castor or Imp? Is Notch required in certain neural lineages and not others. These studies will be of interest in the community of developmental neurobiologists.

Reviewer #2 (Public Review):

Embryonic stem cells extensively proliferate to generate the necessary number of cells that are required for organogenesis, and their proliferation must be timely terminated to allow for proper patterning. Thus, timely termination of stem cell proliferation is critical for proper development. Numerous studies have suggested that cell-extrinsic changes in the surrounding niche environment drive the termination of stem cell proliferation. By contrast, cell-intrinsic mechanisms that terminate stem cell proliferation remain poorly understood. Fruit fly larval brain neuroblasts provide an excellent model for mechanistic investigation of intrinsic control of stem cell proliferation due to the wealth of information on molecular marks, gene functions and lineage hierarchy. Sood et al. conducted a genetic screen to identify genes that are required for the termination of neuroblast proliferation in metamorphosis and found that Notch and its ligand Delta contribute to their exit from cell cycle. They showed that knocking down Notch or delta function in larval neuroblasts allows them to persist into adulthood and remain proliferative when no neuroblasts can be detected in wild-type adult brains. By carrying out a well-designed temperature-shift experiment, the authors showed that Notch is required early during larval development to promote timely exit from cell cycle in metamorphosis. The authors went on to show that attenuating Notch signaling prolongs the expression of temporal identity genes castor and seven-up perturbing the switch from Imp to Syp/E93. Finally, they showed that knocking down Imp function or overexpressing E93 can restore the elimination of neuroblasts in Notch/delta mutant brains.

Overall, the experiments are well conceived and executed, and the data are clear. However, the data reported in this study represent incremental progress in improving our mechanistic understanding of the termination of neuroblast proliferation. Some of the data seem to represent more careful analyses of previously published observations described in the Zacharioudaki et al., Development 2016 paper while others seem to contradict to the results in this study. Gaultier et al., Sci. Adv. 2022 suggested that Grainyhead is required for the termination of neuroblast proliferation in a neuroblast tumor model, and grainyhead is a direct target of Notch signaling. Thus, Grainyhead should be a key downstream effector of Notch signaling in terminating castor and seven-up expression. Identical to Notch signaling, Grainyhead is also expressed through larval development. Grainyhead can function as a classical transcription factor as well as a pioneer factor raising the possibility that temporal regulation of neurogenic enhancer accessibility might be at play in allowing Notch signaling in early larval development to set up termination of castor and seven-up expression in metamorphosis. Diving deeper into how dynamic changes in chromatin in neurogenic enhancers affect the termination of neuroblast proliferation will significantly improve our understanding of termination of stem cell proliferation in diverse developing tissue.

Reviewer #3 (Public Review):

In this study, the authors investigate the effects of Notch pathway inactivation on the termination of Drosophila neuroblasts at the end of development. They find that termination is delayed, while temporal patterning progression is slowed down. Forcing temporal patterning progression in a Notch pathway mutant restores the correct timing of neuroblast elimination. Finally, they show that Imp, an early temporal patterning factor promotes Delta expression in neuroblast lineages. This indicates that feedback loops between temporal patterning and lineage-intrinsic Notch activity fine tunes timing of early to late temporal transitions and is important to schedule NB termination at the end of development.
The study adds another layer of regulation that finetunes temporal progression in Drosophila neural stem cells. This mechanism appears to be mainly lineage intrinsic - Delta being expressed from NBs and their progeny, but also partly niche-mediated - Delta being also expressed in glia but with a minor influence. Together with a recent study (PMID: 36040415), this work suggests that Notch signaling is a key player in promoting temporal progression in various temporal patterning system. As such it is of broad interest for the neuro-developmental community.

Strengths
The data are based on genetic experiments which are clearly described and mostly convincing. The study is interesting, adding another layer of regulation that finetunes temporal progression in Drosophila neural stem cells. This mechanism appears to be mainly lineage intrinsic - Delta being expressed from NBs and their progeny, but also partly niche-mediated - Delta being also expressed in glia but with a minor influence. A similar mechanism has been recently described, although in a different temporal patterning system (medulla neuroblasts of the optic lobe - PMID: 36040415). It is overall of broad interest for the neuro-developmental community.

Weaknesses
The mechanisms by which Notch signaling regulates temporal patterning progression are not investigated in details. For example, it is not clear whether Notch signaling directly regulates temporal patterning genes, or whether the phenotypes observed are indirect (for example through the regulation of the cell-cycle speed). The authors could have investigated whether temporal patterning genes are directly regulated by the Notch pathway via ChIP-seq of Su(H) or the identification of potential binding sites for Su(H) in enhancers. A similar approach has been recently undertaken by the lab of Dr Xin Li, to show that Notch signaling regulates sequential expression of temporal patterning factors in optic lobes neuroblasts (PMID: 36040415), which exhibit a different temporal patterning system than central brain neuroblasts in the present study. As such, the mechanistic insights of the study are limited.