Author response:
We would like to express our gratitude to all three reviewers for their time and valuable feedback on the manuscript. Below, we provide our point-by-point responses to their comments. Additionally, we summarize here the experiments we plan to conduct in accordance with the reviewers' suggestions:
Revision plan 1. To further explore the mechanisms of Notch signaling in the decision of regional EE pattern.
Our observation of EE subtype changes in Notch mutant clones revealed that Notch is required for the specification of Type II EEs, but whether it promotes the generation of Type III EEs is not quite clear. In this revision, we will complete the quantification of Type I and Type III EEs in Notch mutant clones to demonstrate whether Notch signaling participate the determination of these two EE subtypes. Further, we will attempt to combine Notch mutant with different manipulation of WNT and BMP gradients to investigate their interplays.
Revision plan 2. To supplement the global pattern of WNT and BMP gradient along the whole gut.
The levels of WNT and BMP gradients are variable in different gut regions both under normal condition and genetic manipulation, leading to different outcomes of EE subtype composition. To further support our model, we will supply the changes of WNT and BMP gradients along the whole gut after genetic manipulation, and perform semi-quantification of their levels to correlate with EE subtype compositions. Additionally, we will also test the gradient levels at different time point during pupal stage to interpret the establishment of regional identity during the development.
Revision plan 3. To investigate the involvement of apical-basal polarity in the determination of regional EE diversity.
Although we have demonstrated WNT and BMP gradients orchestrate the regional EE identity, but some observations cannot be fully explained by their roles, such as asymmetric expression of CCHa2 in EE pairs from R4b. A potential mechanism is apical-basal polarity, which has been reported to determine cell fate of ISC progenies at pupal stage. We will specifically knockdown or overexpress key genes related to apical-basal polarity in ISCs or EEs to test whether they are involved preliminarily.
Please find our detailed point-by-point responses below.
Public Reviews:
Reviewer #1 (Public review):
This valuable study explores the regulatory mechanisms underlying the regional distribution of enteroendocrine cell subtypes in the Drosophila midgut. The regional distribution of EE cell subtypes is carefully documented, and the data convincingly show that each EE cell subtype has a unique spatial pattern. The study aims at determining how the spatial distribution of EE cell subtypes is established and maintained, and explores the roles of three pathways: Notch, WNT, and BMP. The data show evidence that Notch signaling regulates the subtype specificity, being necessary for the specification of Type II, but not Type I and III EE cell subtype specification. The immunofluorescence data in Figure 3 are convincing, but the analysis is incomplete due to a lack of quantification. How Notch signaling activity relates to the emergence of the regional EE cell patterns remains unclear.
Indeed, the role of Notch signaling in regional EE determination was not fully characterized in this work. As the requirement of Notch activation for the differentiation of enterocytes, introduction of Notch or Delta mutant led to rapid accumulation of ISCs and EEs, making it being a challenge to dive into the details of how EE subtypes were generated. We will try to complete the quantification of Type I and Type III EEs in the Notch mutant clones from different gut regions to figure out whether Notch could influence the specification of these two EE subtypes. Additionally, different from WNT and BMP gradients, Notch signaling can only function locally and is not significantly changed along the whole gut, including Type II EE-enriched R1a and Type I EE-enriched R4b, which implies that function of Notch signaling may can be overridden by the impact of specific combination of WNT and BMP gradients. To test this hypothesis, we will attempt to combine Notch mutant with the activation or inhibition of WNT and BMP signaling since pupal stage, and further examine whether the regional EE identity could be altered, especially in R1a and R4b regions.
As WNT and BMP are known as morphogens, the study explores their expression patterns and their roles in establishing and maintaining the subtype identities. The observed patterns of WNT and BMP are consistent with earlier studies. Manipulation of WNT and BMP pathway activities in intestinal stem cells is shown to have some region-specific effects on specific EE cell subtypes. The overall conclusion that both WNT and BMP have local effects on EE cell subtypes is based on solid evidence. However, the study falls short in achieving its main objective, i.e., to explain the regional subtype patterns by the action of WNT and BMP gradients. Despite displaying a large volume of phenotypic data in Figures 4-7, the study remains incomplete in providing sufficient evidence to support the models shown in Figures 7 M and N. The main challenge is that the reader is provided with a large volume of individual data fragments of selected regions (e.g., Figures 4 and 5) or images of whole midgut without proper quantification (Figure 7). There is not sufficient effort made to display the data in a way that allows observing changes in the global patterns of EE cell subtypes throughout the midgut and compare these patterns with the observed WNT and BMP gradients.
As the variation of WNT and BMP gradients along the whole gut, manipulating these two pathways is not able to align their activation levels in different gut regions. This forced us to analyze the change of each region separately, making it to be a challenge to provide a comprehensive global overview. We will supplement the comprehensive profile of WNT and BMP activity under the manipulation of these two signaling pathways to correlated with the change of EE identity, and also try to perform a semi-quantitative interpretation to further support the model in Figure 7M and 7N.
Reviewer #2 (Public review):
Summary:
By labeling the three major enteroendocrine cell markers - AstC, Tk, and CCHa2-the authors systematically investigated the distribution of distinct EE subtypes along the Drosophila midgut, as well as their emergence via symmetric and asymmetric divisions of enteroendocrine progenitor cells. Moreover, they dissected the molecular mechanisms underlying regional patterning by modulating Wnt and BMP signaling pathways, revealing that these compartment boundary signals play key roles in regulating EE subtype diversity.
Strengths:
This work establishes a solid methodological and conceptual foundation for future studies on how stem cells acquire positional identity and modulate region-specific behaviors.
Weaknesses:
Given that the transcriptional profiles of intestinal stem cells across different regions are highly similar, it is reasonable to hypothesize that the behavior of ISCs and enteroendocrine precursor cells may be regulated non-autonomously, potentially through interactions with enterocytes, which exhibit more distinct region-specific characteristics.
This is a quite complicated point to discuss. Drosophila adult midgut is established by pISCs (pupal ISCs), which arise from AMPs (adult midgut progenitors) in larval midgut. AMPs are encased by PCs (peripheral cells) to be islands, scattered throughout the entire larval midgut by mid L3 stage (Mathur D. et al. Science. 2010). After pupariation, larval midgut is delaminated to become the yellow body and finally meconium in the pupal midgut. Simultaneously, PCs break down and die, allowing AMPs to give rise to the presumptive adult epithelium (generating enterocyte precursors) and the specification of ISCs in the adult midgut (Jiang H, Edgar BA. Development. 2009; Micchelli CA. et al. Gene Expr Patterns. 2011). During the pupal stage, pISCs only proliferate to generate new ISCs and EE lineages, while adult enterocytes start to appear after eclosion (Takashima S. et al. Dev Biol. 2011). This rules out the possibility that the interaction with enterocytes regulates regional ISC identity during pupal stage.
However, whether AMPs already acquire the regional identity during larval stage, and whether pISCs interact with enterocyte precursors at pupal stage, are not quite clear. Our study revealed that pISCs can be influenced by WNT and BMP gradients to acquire regional identity, and further establish regional EE diversity. The change of WNT and BMP gradients during the metamorphosis will be supplemented in revision. While WNT and BMP signaling ligands are provided by muscles and adult enterocytes, and even other surrounding tissues, to regulate regional ISC identity, which indicates that non-autonomous mechanisms indeed exist.
Reviewer #3 (Public review):
Summary:
The authors aimed to elucidate the mechanisms underlying the regional patterning of enteroendocrine cell (EE) subtypes along the Drosophila midgut. Through detailed immunohistochemical mapping and genetic perturbation of Notch, WNT, and BMP signaling pathways, they sought to determine how extrinsic morphogen gradients and intrinsic stem cell identity contribute to EE diversity.
Strengths:
A major strength of this work is the meticulous regional analysis of EE pairs and the use of multiple genetic tools to manipulate signaling pathways in a spatiotemporally controlled manner. The data robustly demonstrate that WNT and BMP signaling gradients play key roles in specifying EE subtypes and division modes across different gut regions.
Weaknesses:
However, the study does not fully explore the mechanistic basis for the region-specific dependence on Notch signaling. Additionally, while the authors propose that symmetric divisions occur in R1a and R4b, the observed heterogeneity in CCHa2 expression within AstC+ pairs in R4b suggests that asymmetric mechanisms may still be at play, possibly involving apical-basal polarity as previously reported.
As previously mentioned, we acknowledge that the role of Notch signaling in regional EE determination remains further exploration. We will supplement the quantification of Type I and Type III EEs in Figure 3 and Figure S4, and further combine Notch mutant with activation or inhibition of WNT and BMP signaling to test whether they have any interplays, especially in R1a and R4b.
Apical-basal polarity has been reported to determine the precise segregation of Pros to control ISC number and cell fate at the pupal stage (Wu S. et al. Cell Rep. 2023). During this time, generation of regional EEs are completed and may also be affected except for the influence of Notch, WNT and BMP pathways. Therefore, the apical-basal polarity is quite a potential mechanism to induce asymmetric cell division in R4b, which we will perform experiments to test.
Appraisal of achievements:
The authors successfully achieve their aims by providing a compelling model in which intercalated WNT and BMP gradients regulate EE subtype specification and EEP division modes. The genetic data strongly support the conclusion that these pathways are central to establishing regional EE diversity during pupal development.
