An evolutionarily conserved Hox-Gbx segmentation code in the rice coral Montipora capitata

Reviewer #1 (Public review):

Summary:

The manuscript of He et al. compares the roles of Hox/Gbx genes between the well-established anthozoan model, the burrowing sea anemone Nematostella, and the new scleractinian model Montipora. The authors show staggered expression of Anthox6a.1, Anthox8 and Gbx of the Montipora larva and argue that their BMP-dependent expression is responsible for the segmentation of the endomesoderm, just like they have previously demonstrated in Nematostella (despite some differences in the timing, formation of extra mesenteries, etc). The authors posit that Hox/Gbx-dependent segmentation of the endomesoderm represents an ancestral anthozoan trait. The study addresses a remarkably interesting question, but it has several important shortcomings, which the authors should try to rectify.

Strengths:

The authors introduce a new scleractinian model Montipora and present interesting data on the composition of its compact Hox cluster, its embryonic and larval development, metamorphosis, and segmentation. They also show staggered expression of Gbx, Anthox6a.1, and Anthox8, which is suggestive of their involvement in the partitioning of the gastrodermis of the polyp.

Weaknesses:

He et al. claim that Gbx and Hox genes are responsible for the segmentation of the directive axis in Montipora based on expression patterns of these genes before the onset of segmentation. In the absence of functional analyses, this claim (although likely correct) is not supported. Moreover, the authors do not show that staggered Gbx and Hox gene expression correlates with the position of the segment boundaries.

The authors use two inhibitors of BMP signaling and show that segmentation is lost in the treated animals. However, they do not provide controls, which would show that the effect of the treatment is specific to the loss of BMP function. Moreover, their transcriptomic analyses suggest that the whole BMP signaling system in Montipora is wired completely differently than in Nematostella, but they do not acknowledge and discuss this striking difference. If true, this is a very interesting result, but it requires thorough validation.

Reviewer #2 (Public review):

Building on their detailed dissection of the role of Hox-Gbx genes in endomesodermal segmentation in Nematostella, He and colleagues attempt to understand the evolutionary conservation of this process in anthozoans. In a move that should be congratulated, the authors perform this work in the coral M. capitata, a species that is not well established in the lab. The authors show convincing expression data using both RNAseq and in-situ hybridization and discover the conserved expression of Hox-Gbx genes preceding the segmentation of the enodmesoderm. The authors further attempt to understand whether BMP signalling is playing a role in this process and present data that certainly points to this being the case.

Strength:

The overall quality of the data is very high and the authors show very convincing expression data for the Hox-Gbx genes as well as putting forward a well-thought-out hypothesis for segment evolution.

Weakness:

There are a number of weaknesses in the paper which I believe can be easily addressed:

(1) The authors in many cases claim to have provided functional evidence for the role of Hox-Gbx genes in M. capitata. This is not, however, the case, and although the expression data along with their previous work in Nematostella make their claims very likely I still believe it is necessary to set a higher bar for claiming to understand function. In the abstract, for example, they claim: "These findings demonstrate the existence of a functionally conserved Hox-Gbx module....", something which is not substantiated by the data presented. At the end of the introduction, they say they "systematically interrogate the molecular functions of Hox-Gbx genes" (line 75) which again is not what is presented in the manuscript. Finally, on line 289-291 the authors state: "Taken together, our findings strongly suggest that the heterochronic deployment of a conserved Hox-Gbx module contributes to the divergent adult body plans observed between Edwardsiidae and other anthozoans." I would remove "Strongly" given the absence of functional data. There are also other examples where functional understanding is implied and I would suggest the authors tone this down throughout the manuscript.

(2) On Line 185, the authors state "To determine the function of the Hox-Gbx network in M.capitata segmentation..." when introducing their BMP experiments. I would reword this since they are looking at BMP signalling and do not look directly at Hox-Gbx function.

(3) Although the BMP inhibitor experiments are very interesting I think there is a lack of basic understanding of BMP signalling in this system. Where are the BMP components expressed and how would this match with the hypothesis derived from the data? The authors present some expression patterns in Figure S3 but do not discuss them. In addition, the authors do not show pSMAD staining etc, and do not validate that the inhibitors have an effect on this. I entirely understand the difficulties in doing such experiments in a system like this and would not suggest the authors should now do them but an acknowledgment of this in the discussion would be very welcome.

(4) In both lines 88 and 294 the authors talk about the mechanism of gastrulation. It is not clear to me how they infer this from the figure. If the authors could include some more high-resolution images that show this it would be very helpful and interesting.

(5) On line 169/170 the authors state that two Anthox6 paralogs, McAnthox6 and McAnthox6.1, were specifically expressed at the time of settlement. This is not what I see in the images. I see that McAnthox6 is expressed at 14 hpf more strongly than at the later time point. The authors should clarify this point.

(6) On lines 259-261 the authors state "How temporally and spatially coordinated gene expression can be achieved in this scenario remains an interesting and open question." This seems like a strange statement to include given that they have shown that there is no spatial and temporal collinearity in cnidarians. Surely it is not an open question to ask how it would work if there is none. I would simply remove this.

(7) The authors should cite the sources of information contained in Fig. S2 including how orthology was assigned.

Reviewer #3 (Public review):

Summary:

The authors analyze the expression of a series of genes from the Hox/Gbx family of transcription factors in the settling larva of the rice coral Montipora capitata. The first achievement of the work is developing a protocol for artificial induction of settlement in this species. In the synchronized settlers, the authors were able to follow the sequence of the subdivision of the body cavity to form individual cavities separated by mesenteries. This process has been previously studied in the starlet sea anemone, Nematostella vectensies, and this same group showed that there is a spatio-temporal sequence of expression of genes from the Hox/Gbx group, reminiscent of the sequence of Hox genes in bilaterians. The authors now repeat this analysis with orthologous genes in Montipora, and demonstrate a similar pattern. Finally, they manipulate the BMP pathway and demonstrate that in the absence of BMP signaling, the subdivision of the gastric cavity is abrogated.

Strengths:

The authors have developed a new experimental system for embryological work on cnidarians, where only a handful of systems are available. They identified orthologs of a number of homeobox genes and tested their expression. There is a detailed description of the sequence of the formation of the mesenteries, which differs from that of Namatostella, raising interesting questions about the evolution of mesentery number and the homology of mesenteries.

Weaknesses:

The in situ hybridization experiments describing the expression of the Hox/Gbx genes are not as clean and sharp as could be hoped for. This is evidently a limitation of the system. The discussion of the evolution of mesentery number does not really give new insights into the question (although just raising the discussion is interesting in its own right).