Spinal neural tube formation and regression in human embryos

Reviewer #1 (Public Review):

Summary:
The authors analyzed 102 human embryos in order to address outstanding questions about human lower spinal development and secondary neural tube formation. Through whole embryo imaging and histologic analysis, they provide exceptional quantification of the timing of posterior neuropore closure, rate of lower spinal somite formation, and formation and regression of the human "tail." Their analysis also provides convincing qualitative evidence of the cellular and molecular mechanisms at play during lower spinal development by identifying the presence of caspase-dependent programmed cell death and the dynamic expression of FGF8/WNT3A within the elongating embryo. Interestingly, they identify multiple polarized lumens within the site of secondary neural tube formation and add a solid argument for the mode of formation of this structure; however, in its current state, the evidence for a conclusive morphogenetic mechanism remains elusive. Finally, the authors provide a substantial review of the existing publications related to human lower spinal development, creating an excellent reference and demonstrating the importance of continuing to utilize each of these precious samples for furthering our understanding of human development.

Strengths:
This manuscript provides an excellent window into the key morphogenetic events of human caudal neural tube formation. Figures 1 and 2 provide beautiful images and quantification of the developmental events, enabling comparison to models that are currently in use, including model organisms and the developing spinal organoid field. The characterization of somite development and later regression is particularly important.

Next, the authors addressed current questions regarding the molecular pathways present during the elongation of the embryo and later regression of the tail structure. The in situ hybridization experiments in Figures 5 and 6 demonstrate important evidence for a maintained neuromesodermal progenitor pool of stem cells that promote axial elongation. Additionally, the identification of caspase-dependent cell death within the human tail provides an explanation for the mechanism of this regression, especially given the notable lack of presence of any gross necrosis.

Finally, as mentioned above, the non-trivial collection and review of the existing human secondary neural tube and body formation literature is an important tool and organizes and synthesizes ~ 100 years of observations from precious human samples.

Weaknesses:
While there are no glaringly incorrect claims from the authors, several of the conclusions could benefit from a form of quantification to support their observations:

1. The identification of the proximal to distal degeneration of the tailgut within the human tail is difficult to distinguish with the current images present in Figure 3. A picture within a picture of the area containing the tail gut could be provided to prominently demonstrate the cellular architecture. Additionally, quantification of the localization of apoptosis would strongly support this observation, as well as provide a visualization of the tail's regression overall. For example, a graph plotting the number of apoptotic cells versus the rostral to caudal locations of the transverse sections while accounting for the CS stage of each analyzed embryo could be created; this could even be further broken down by region of tail, for example, tailgut, ventral ectodermal ridge, somite, etc.

2. The identification of the mode of formation of the secondary neural tube is probably the most interesting question to be addressed, however, Figure 7's evidence is not completely satisfying in its current form. While I agree that it is unlikely that multiple polarization foci form within the most caudal part of the tail and coalesce more rostrally, I am equally unsure that a single polarization would form rostrally and then split and re-coalesce as it moves caudally, as is currently depicted by 7B.

Multiple groups have recently shown the influence of geometric confinement on neuroectoderm and its ability to polarize and form a singular central lumen (Karzbrun 2021, Knight 2018), or the inverse situation of a lack of confinement resulting in the presence of multiple lumens. The tapering of the diameter of the tail and its shared perimeter and curvature with the polarization bears a striking resemblance to this controlled confinement. An interesting quantification to depict would include the number of lumens versus the transverse section diameter and CS stage to see if there is any correlation between embryo size and the number of multiple polarizations. Anecdotally, the fusion of multiple polarizations/lumens tends to occur often in these human organoid-type platforms, while splitting to multiple lumens as the tissues mature does not. Other supplements to Figure 7 could include 3D renderings of lumens of interest as depicted in Catala 2021, especially if it demonstrates the re-coalescence as seen in 7B.

The non-pathologic presence of multiple polarizations in human tails compared to the rodent pathogenic counterpart is interesting given that rodents obviously maintain this appendage while it is lost in humans.

1. Of potential interest is the process of junctional neurulation describing the mechanistic joining of the primary and secondary neural tube, which has recently been explored in chick embryos and demonstrated to have relevance to human disease (Dady 2014, Eibach 2017, Kim 2021). While it is clear this paper's goal does not center on the relationship between primary and secondary neurulation, such a mechanism may be relevant to the authors' interpretation of their observations of lumen coalescence. I wonder if the embryos studied provide any evidence to support junctional neurulation.

Reviewer #2 (Public Review):

Summary:
This study utilizes a large series of neurulation human embryos to address several questions about the similarities and differences between human neurulation and model systems such as the chicken and rodent.

Strengths:
The number of specimens utilized for the analysis provides robustness to the findings.

Weaknesses:
It is not clear how the gestational age of the specimens was determined or how that can be known with certainty. There is no information given in the methods on this. With this in mind, bunching the samples at 2-day intervals in Figure 1J will lead to inaccuracies in assessing the rate of somite formation. This is pointed out as a major difference between specimens and organoids in the abstract but a similar result in the results section. The data supporting either of these statements is not convincing.

Whenever possible, give the numbers of specimens that had the described findings. For example, in Figure 2C - how many embryos were examined with the massive rounded end at CS13? Apoptosis in Figures 3 and 4?

For Figure 2I-K, it would be informative to superimpose the individual data points on the box plots distinguishing males from females, as in Figure 1I.

Is it possible to quantitate apoptosis and proliferation data?

The Tunel staining in Figure 3 is difficult to make out.

Additional improvements to the presentation of figures, writing, and quantization of results are suggested.