Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination

Reviewer #1 (Public Review):

Summary:
This describes the molecular identity of the intermediate status of cranial neural crest cells (NCCs) during the initial delamination process. Taking advantage of single-cell RNA seq, the authors identify new populations of cells during EMT characterized by a specific set of gene expressions, including Dlc1. Promigratory cranial NCCs differentiate through different trajectories depending on their cell cycle phases but converge into a common progenitor, then differentiate into mesenchymal cells expressing region-specific genes.

Strengths:
Single-cell RNA seq data convincingly support what the authors claim. This is the first time to identify intermediate states between premigratory and migratory cranial NCCs. Silencing one of the marker genes, Dlc1, reduces the migratory activity of cranial NCCs. These findings deepen our understanding of the mechanism of EMT in general.

Weaknesses:
Common and specific features between cranial and trunk NCCs could be described/discussed in-depth. Phenotypic relations between the reduction of delamination and defects found in Dlc1 mutant mice can be discussed.

Reviewer #2 (Public Review):

Zhao et al., focus on mechanisms through which cells convert from epithelium to mesenchyme and become migratory. This phenomenon of epithelial-to-mesenchymal transition (EMT) occurs during both embryonic development and cancer progression. During cancer progression, EMT seemingly includes cells at intermediate states as defined by the combinatorial expression of epithelial and mesenchymal markers. However, the importance of these markers and the role of these intermediate states remains unclear. Moreover, whether EMT during development also involves equivalent intermediate cell states is not known. To address this gap in knowledge, the authors devise a strategy to identify and characterize changes that an embryonic population of cells called the cranial neural crest undergo as they delaminate from the neuroepithelium and become a highly migratory population of mesenchymal cells that ultimately give rise to a broad range of derivatives.

To isolate and study the neural crest, the authors use embryos collected at E8.5 from two transgenic mouse lines. Wnt1-Cre;RosaeYFP labels Wnt1-positive neuroepithelial cells in the dorsolateral neural plate, which includes pre-migratory neural crest that resides in the dorsal neuroectoderm and neural plate border before induction (as well as some other lineages). Mef2c-F10N-LacZ leverages a neural crest cell-specific enhancer of Mef2c to control LacZ expression in the predominantly migratory neural crest. This dual genetic approach that allows the authors to distinguish and compare pre-migratory and migratory neural crest cells is a strength of the work. However, one potential weakness needing to be addressed is that some workers (e.g., Lewis et al., 2013) have reported phenotypic effects of Wnt1-Cre transgene expression including ectopic Wnt pathway activation, abnormal neuroepithelial development, and increases in CyclinD1 expression and cell proliferation. The authors should discuss the extent to which the results of their study were or were not influenced by these potentially confounding effects, especially since Wnt canonical signaling is known to regulate the G1/S transition and promote delamination of the neural crest.

To assay for the differential expression of genes involved in the EMT and migration of cranial neural crest, the authors perform single-cell RNA sequencing (scRNA-seq) using current methods. A strength is a large sample size per mouse line, and relatively high numbers of single cells analyzed. The authors identify six major cell/tissue types present in mouse E8.5 cranial tissues using known markers, which they then segregate into a cranial neural crest cluster using a well-reasoned bioinformatic strategy. The cranial neural crest cluster contains pre-migratory and migratory cells that they partition further into five subclusters and then characterize using the differential expression and combinatorial patterns of neural crest specifier genes, markers of pre-migratory neural crest, markers of early versus late migratory neural crest, markers of undifferentiated versus differentiated neural crest, tissue-specific markers, and region-specific markers. One weakness is that there is no attempt to map potential novel genes and/or pathways that also distinguish these clusters.

The authors then go on to subdivide the five cranial neural crest subclusters into almost two dozen smaller subclusters, again using the combinatorial expression of known markers (e.g., neural crest genes, cell junction genes, and cell cycle genes). A weakness is that the marker analysis and accompanying interpretation of the results rely heavily on the purported roles of different genes as described in the published work of others, which potentially introduces some untested assumptions and a bit of hand-waving into the study. Moreover, the limited correlation between mRNA and protein abundance for cell cycle markers is well documented in the literature but the authors rely heavily on gene expression to determine cell cycle status. Even though the authors add a compelling Edu/pHH3 double-labeling experiment and cell cycle inhibition studies, the work would be strengthened by including some analysis of protein expression to see if the cell cycle correlations hold up. Nonetheless, the subcluster and cell cycle analyses lead the authors to conclude that there are a series of intermediate cell states between neural crest EMT and delamination, and that cell cycle regulation is a defining feature and necessary component of those states. These novel findings are generally well supported by the data.

To test if there are spatiotemporal differences in the localization of neural crest cells during EMT in vivo, the authors apply a cutting-edge technique called signal amplification by exchange reaction for multiplexed fluorescent in situ hybridization (SABER-FISH), which they validate using standard in situ hybridization. The authors select specific marker genes that seem justified based on their scRNA-seq dataset, and they generate a series of convincing images and quantitative data that add valuable depth to the story.

As a functional test of their hypothesis that one of the genes indicative of an EMT intermediate stage (i.e., Dlc1) is essential for neural crest migration, the authors use a lentivirus-mediated knockdown strategy. A strength is that the authors include appropriate scramble and cell death controls as part of their experimental design. However, a weakness is that the authors do not justify why they chose a knockdown strategy, which has its limitations including its systemic injection into the amniotic cavity, its likely global and more variable effects, and its need to be conducted in culture. Why the authors did not instead use a Wnt1-Cre-mediated deletion of Dlc1, which would have been "cleaner" and more specific to the neural crest, is not clear (maybe so they could specifically target different Dcl1 isoforms?). Also, the authors use Sox10 as a marker to count neural crest cells, but Sox10 may only label a subset of neural crest cells and thus some unaffected lineages may not have been counted. The authors should mention what is known about the regulation of Dcl1 by Sox10 in the neural crest. Although the data are persuasive, a second marker for counting neural crest cells following knockdown would make the analysis more robust. Can the authors explain why they did not simply use the Mef2c-F10N-LacZ line and count LacZ-positive cells (if fluorescence signal was required for the quantification workflow, then could they have used an anti-beta Galactosidase antibody to label cells)?

Overall, this is a first-rate study with many more strengths than weaknesses. The authors generate high-quality data, and their interpretations are reasonable and balanced. Another strength is the writing, which is clear and well organized, and the figures (including supplemental), which are excellent and provide unambiguous visualization of some very complex data sets. The methods are state-of-the-art and are effectively executed, and they will be useful to the broader cell and developmental biology community. The work contains well-substantiated findings and supports the conclusion that EMT is a highly dynamic, multi-step process, which was previously thought to be more-or-less binary. Such findings will alter the way the field thinks about EMT in neural crest and the work will likely serve as an important example alongside cancer metastasis.

Reviewer #3 (Public Review):

Summary:
Zhao et al. address the question of whether intermediate states of the epithelial-to-mesenchymal transition (EMT) exist in a natural developmental context as well as in cancer cells. This is important not only for our understanding of these developmental systems but also for their development as resources for new anti-cancer approaches. Guided by single-cell RNA sequencing analysis of delaminating mouse cranial neural crest cells, they identify two distinct populations with transcriptional signatures intermediate between neuroepithelial progenitors and migrating crest. Both clusters are intermediate spatially and actively cycling, with one in S-phase and one in G2/M. They show that blocking progression through S phase prior to the onset of delamination and knockdown of intermediate state marker Dlc1 both reduce the number of migratory cells that have completed EMT. Overall, the work provides a modern take and new insights into the classical developmental process of neural crest delamination.

Strengths:
• Deep analysis of the scRNAseq dataset revealed previously unappreciated cell populations intermediate between premigratory and migratory crest.
• The observation that delaminating/intermediate neural crest cells appear to be in S or G2/M phase is interesting and worth reporting, though the ultimate significance remains unclear, given that they do not make distinct derivatives depending on their cycle state.
• The authors employ new methods for multiplex spatial imaging to more accurately define their populations of interest and their relative positions.
• The authors present evidence that intermediate state gene Dlc1 (a Rho GAP) is not just a marker but functionally required for neural crest delamination in mice, as previously shown in chicken.

Weaknesses:
• Similar experiments involving blockade of cell cycle progression and Dlc1 dose manipulation were previously performed in chick models, as noted in the discussion. The newly-defined intermediate states give added context to the results, but they are not entirely novel.
• The putative intermediate cells differentially express mRNAs for genes involved in cell adhesion, polarity, and protrusion relative to bona fide premigratory cells (Fig. 2E). This is persuasive evidence, but only differentially expressed genes are shown. Discussing those markers that have not yet changed, e.g. Cdh1 or Zo1 (?), would be instructive and help to clarify the order of events.
• It is unclear whether the two putative intermediate state clusters differ other than their stage of the cell cycle. Based on the trajectory analysis in Fig. 3C-D, the authors state that these two populations form simultaneously and independently but then merge into a single population. However, without further differential expression, it seems more plausible that they represent a single population that is temporarily bifurcated due to cell cycle asynchrony.
• The authors do not present an in-depth comparison of these neural crest intermediate states to previously reported cancer intermediate states. This analysis would reveal how similar the signatures are and thus how extrapolatable these and future findings in delaminating neural crest are to different types of cancer.
• Lines 265-289 (Fig. 4): The aphidicolin treatments appear to have been started before NC delamination begins in earnest, so the fact that there are any migratory SOX10+ neural crest in the treated embryos at all indicates that progression through S-phase is not explicitly required for delamination. The authors surmise that the successfully delaminated cells may instead have been in G2/M phase (perhaps representing cluster 10') already at the start of treatment and thus able to progress through EMT, while S phase intermediate and true premigratory cells were not. This is plausible. However, the reduction in SOX10+ cells may be in part or wholly attributable to inhibition of proliferation AFTER delamination. Showing that there are premigratory NCCs in G2/M at ~E8.0 would bolster the argument that this population is present from the earliest stages.