Reviewer #2 (Public Review):
The manuscript describes the development of a new digital 3D tool to monitor spatial and temporal changes in the neural tube, using hindbrain morphogenesis as a proof of concept tissue. The conclusions of the manuscript are supported by the results and the quality of the data is excellent.
Strengths
1. The data and results of the manuscript have been meticulously prepared and support the author's conclusions.
2. Open-access, novel, and user-friendly protocols for digitalizing spatial and temporal changes of tissue growth are in demand and will clearly contribute to the relevant scientific communities
3. Inclusion of 3D temporal information to the already existing 3D-spatial atlases is missing. The new methodology developed in this manuscript will open the door for collecting valuable information (which is currently limited) on normal and perturbed brain development in time and space.
4. The quality of the presented data is very good.
5. The development of the hindbrain is a fascinating process that is less studied in comparison to higher brain areas. As proper hindbrain development is the foundation upon which the brainstem will later form, novel knowledge on hindbrain morphogenesis and neurogenesis is of clear importance.
Weaknesses
1. Compared to other methodology-based papers on brain image analysis (i.e., Chow et al., 2020; Jaggard et al., 2020; Kenney et al., 2021; Ronneberger et al., 2012; Tabor et al., 2019; Dsilva et al., 2015; Fernandez and Moisy, 2020), the manuscript is a bit narrow in its overall information. For instance, this manuscript uses different transgenic lines to quantify cells of different neuronal subpopulations at several time points to (elegantly) show their differentiation dynamics. Yet, the magnifications are very low so that the provided data is an overview of the entire hindbrain without tracing the cell's behavior at a much higher 3D resolution. This is at variance from some of the above-cited papers which imaged cell domains at much higher magnifications.
2. The temporal component (emphasized by the authors as their main novelty) did not integrate data from ongoing time-lapse imaging of color-converted cells, (although expected when reading the Introduction and Abstract). Rather, the analysis was based on a comparison of fixated brains at 3 different stages from different fishes. Without diminishing the importance of this comparison (which was elegantly done), putting the temporal-based analysis in the forefront of the methodology is a bit misleading, as it occupies only a small part of the manuscript.
3. The paper does not take the newly developed protocols one step further to serve as a proof-of-concept study by using fish with normal or aberrant neurogenesis. This diminishes the powerfulness of the suggested technology. Such types of analyses have been provided in some other papers which developed recent 3D digital atlases of the brain, where the technologies were utilized to answer developmental/behavioral questions.
4 . In general, the many efforts used in this study to develop the imaging technologies did not seem to yield a significant amount of novel information, when compared to more standard imaging techniques (that the authors present). Hence, more substantial data is needed to convince how the DAMAKER is a "game-changer" in the field of neural development.
