The tuxedo sea urchin Mespilia globulus: A fast-developing and tractable model for genomic and developmental biology

Reviewer #1 (Public review):

Summary:

In this manuscript, Matar et al. introduce Mespilia globulus, the tuxedo sea urchin, as a new genomically-enabled model for echinoderm developmental biology. The authors establish a closed life-cycle culture system in a land-locked aquarium facility, demonstrate that key experimental techniques (hybridization chain reaction labelling and CRISPR/Cas9 gene knockout) are tractable in this species, and report chromosome-scale genome assemblies for two colour morphs and both sexes. Using these resources, they compare genome architecture and gene family evolution across sea urchin species, and investigate the genomic basis of sex determination.

Strengths:

The central motivation for this work is well justified: the long larval and juvenile periods of established sea urchin models such as Strongylocentrotus purpuratus have long limited the study of post-metamorphosis and adult biology, and M. globulus reaches metamorphosis in around two weeks and sexual maturity within four to six months, a substantial acceleration. The husbandry and life-cycle data are thorough, and the demonstration that hybridization chain reaction staining and CRISPR/Cas9 knockout both work as expected in this species convincingly establishes its experimental tractability. The two chromosome-scale genome assemblies are of high quality (BUSCO completeness above 99%, 21 chromosome-scale scaffolds consistent with other sea urchins), and the comparative synteny and gene family analyses are carefully constructed, drawing on a solid phylogenomic framework (CAFE-based gene family turnover analysis across six echinoderm species). The authors' finding that M. globulus has fewer duplicated genes in the gene repertoire relative to other camarodont urchins is a genuinely useful observation for researchers choosing a model system for functional genetics, since fewer paralogues should simplify interpretation of knockout phenotypes.

Weaknesses:

Some claims in the manuscript would benefit from additional supporting detail.

(1) The efficiency of the CRISPR/Cas9 knockout is illustrated qualitatively, but no sample size or penetrance value is reported, making it difficult for readers to judge how robust or reproducible this result is.

(2) The gene annotation is reported to have complete PFAM domain coverage for only 75% of predicted genes, but no independent completeness metric (such as BUSCO scored against the annotated gene set rather than the assembly) is provided, leaving open whether the remaining genes are genuinely novel, partial models, or annotation artefacts.

(3) Finally, at the time of review the NCBI BioProject accession cited for the genome and sequencing data (PRJNA1477966) could not be located, and it is not clear from the text whether this accession, once available, will include the gene annotation and RNA-seq datasets in addition to the raw genomic sequencing reads.

In summary, the authors achieve their stated aim of establishing M. globulus as a tractable, fast-developing echinoderm model, and the genomic and experimental resources presented support this conclusion. The comparative genomic conclusions - conservation of ancestral chromosome linkage groups, absence of a heteromorphic sex chromosome, and a comparatively low rate of gene family expansion - are well supported by the data shown, though some of the finer-grained claims (knockout efficiency, annotation completeness) require some clarification. Given the scarcity of tractable models for post-metamorphosis and adult echinoderm biology, this resource is likely to be of real value to the field, provided the genomic and transcriptomic data are made fully and clearly accessible to the community.

Reviewer #2 (Public review):

This manuscript introduces the tuxedo sea urchin, Mespilia globulus, as a new experimental model for developmental, reproductive and genomic biology. The authors establish culture methods that permit completion of the life cycle in a landlocked aquarium facility, demonstrate the applicability of developmental biology tools including HCR-FISH and CRISPR/Cas9-mediated gene disruption, generate chromosome-scale genome assemblies from two color morphs and both sexes, investigate potential sex determination mechanisms, and compare genome organization and gene family evolution with other sea urchin models. The authors conclude that M. globulus combines a relatively rapid life cycle with genomic tractability and therefore represents a valuable addition to the growing repertoire of genetically accessible sea urchin model systems.

Overall, I found this to be a strong and timely contribution that is well suited for the Tools and Resources category of eLife. The authors provide a comprehensive suite of resources, including husbandry protocols, genomic resources, developmental staging information, and proof-of-principle functional manipulations. The manuscript appropriately places M. globulus in the context of established sea urchin models, particularly Lytechinus pictus, and clearly argues that the new species is complementary rather than a replacement for existing systems. Given the increasing importance of genetically tractable echinoderm models, the development of an additional species that can be maintained and bred in closed aquarium systems is of considerable value to the field.

The evidence supporting the establishment of this model system is convincing, with multiple complementary datasets including life-cycle culture methods, genome assemblies, gene expression analyses, and gene-editing experiments.

My concerns primarily relate to two broader issues that should be addressed, followed by several specific comments.

(1) Genetic Background and Aquarium Trade Populations: A central argument of the manuscript is that M. globulus is attractive as a laboratory model because it is widely cultured in the aquarium trade and may exhibit reduced genetic variability due to captive propagation.

The manuscript states:

"M. globulus is a popular species in the aquarium industry and has excellent properties in tropical aquariums where it has been bred for many years in farming operations that reduce genomic heterogeneity..." (lines 96-98) and later:

"Captive breeding may also lower genetic variability compared to wild-caught individuals..." (lines 389-390).

However, the manuscript does not provide sufficient information to evaluate these claims. Several important questions remain unresolved:

(1) How genetically representative are the sequenced individuals relative to natural populations?

(2) What is known about the provenance and breeding history of the aquarium trade stocks used in this study?

(3) Are these animals derived from a small number of founder populations?

(4) Is there evidence for substantial inbreeding or genetic bottlenecks within commercial brood stocks?

(5) How similar are commercially available animals from different vendors and geographic sources?

These questions are important for both practical and biological reasons. From a practical perspective, researchers wishing to adopt M. globulus need to know whether animals purchased from aquarium suppliers are expected to resemble those analyzed here. From a biological perspective, reduced diversity or founder effects could influence genome assembly characteristics, heterozygosity estimates, gene family analyses, developmental traits, or responses to experimental manipulation.

Even if little information is currently available, the manuscript should explicitly discuss these uncertainties and provide available information regarding stock origin, aquaculture practices, and potential differences between captive and natural populations. A clear discussion of potential limitations would significantly strengthen the manuscript.

(2) Presentation and Interpretation of HCR and Phalloidin Data: Although the HCR and phalloidin experiments are not central to the major conclusions of the paper, they serve as important demonstrations of experimental tractability. At present, however, the presentation of these data is not fully convincing.

The HCR images show detectable signal, but the expression domains are only minimally documented. The manuscript states that expression patterns are consistent with known functions of Nodal and Notch signaling, yet the figures do not sufficiently guide readers to these conclusions (especially readers not familiar with sea urchin development). The signal is relatively diffuse and weak in some panels, and there is little annotation explaining exactly which embryonic territories are expressing the genes of interest. For readers without extensive sea urchin developmental biology expertise, it can be difficult to assess the validity and biological significance of the observed expression patterns.

Similarly, the phalloidin-labeled images provide limited anatomical information because the larvae are largely not labeled. I recommend:

(1) Adding labels identifying relevant embryonic regions and structures.

(2) Including arrows or overlays indicating key expression domains.

(3) Providing higher-magnification insets of relevant regions.

(4) Including selected optical sections rather than relying exclusively on 3D projections.

(5) Identifying known larval muscle groups in the phalloidin images.

(6) Improving image contrast and figure annotation where possible.

These changes would substantially strengthen the claim that established developmental biology methods are readily transferable to M. globulus.

Reviewer #3 (Public review):

Summary:

Omar Matar and colleagues describe how they successfully closed the life cycle of the tropical tuxedo urchin Mespilia globulus in a small, closed aquarium system. Its capacity to flourish under closed culturing conditions sets this sea urchin apart from most echinoderms and indeed most marine invertebrates, which require high-quality flow-through seawater (i.e. coastal access and a flow-through aquarium system). As M. globulus reaches sexual maturity in captivity in 4-6 months, which is shorter than other sea urchins, it opens the potential for transgenerational studies.

Matar et al. have developed a suite of molecular and genomic techniques and resources, including Cas9-mediated gene knockdown and chromosome-scale genome assemblies for both sexes and colour morphs. These strong aquaculture and genomics platforms suggest that M. globulus can provide insights into aspects of the sea urchin life cycle and history that would be difficult to study in other sea urchin models, which historically have been focused on embryogenesis.

Strengths:

This manuscript announces M. globulus as a novel, genome-enabled echinoderm model that can be cultured in a closed system with a few hundred litres of 23/24oC artificial seawater. This allows for experimental analysis of parts of the sea urchin life cycle that are not particularly tractable in other sea urchins, including the developmental biology of metamorphosis, symbiont interactions and immunity through the life cycle, post-settlement biomineralisation, and sex determination. The capacity to knock out embryonic/larval genes using CRISPR/Cas9 and visualise localised embryonic and larval gene expression using HCR are consistent with claims M. globulus can contribute novel insights into basic and applied (aquaculture) biology of sea urchins and echinoderms.

Weaknesses:

The general weakness of this manuscript is that the authors do not explain aspects of their study in enough detail (e.g. a single figure - Figure 2 - presents results of analysis of normal development, HCR and Cas9 knockdowns). This manuscript provides limited methodological detail about the culture system and the analysis of gene expression. Here are a few suggestions on how to improve the manuscript:

First, the authors should provide a thorough description of the methods used to cultivate and maintain M. globulus. This should include further details about the closed aquarium system; a schematic of the system would be insightful. Basic details about husbandry are needed, including (i) stocking densities of adults, embryos/larvae, postlarvae/juveniles; (ii) frequencies of level and water changes/top-ups; (iii) feeding regime at all phases of the life cycle (amount/sea urchin, post-feeding cleaning, etc.). These and other details are essential for the uptake of this model system. This documentation would provide the foundation for future improvements, which include shortening the time to acquire larval competence and sexual maturity, and improving and standardising larval settlement.

Second, the description of the procurement and analysis of mRNA is brief, unreferenced and reads as protocols used for an established model species (e.g. what is PFA in this case - the concentration of paraformaldehyde and the buffer can vary markedly between organisms and life stages). Even the RNA extraction protocols can vary between and within species. For instance, highly pigmented tissues tend to be more difficult to procure useable RNA. The HCR analysis, which is also scantily described, is restricted to embryonic and larval stages. Given the emphasis on the capacity of the M. globulus system to analyse all phases of the life cycle, it would be good to know if HCR can be performed on settled postlarvae, juveniles and adult tissues.

Third, the authors should consider dividing Figure 2, which consists of confocal images of normal development, HCR results and CRISPR/Cas9 knockout results, into three separate figures that explore these studies separately. A figure on normal development could, for instance, include documentation of metamorphosis, with a suite of images of postlarval stages. A figure documenting HCR could be expanded to include more stages, higher magnification images and other genes. A figure on the Cas9 knockdown of a PKS gene can provide details on the normal expression of this gene using HCR and possibly qRT-PCR.

Fourth, there should be consideration of providing more characterisation about the protein-coding genes comprising the chromosomal region (Chr. 4) that has marked differences between sexes. This could go beyond Supplementary Table 4 and Supplementary Figure 5B, and include analysis of expression in adult tissues (this would be enhanced by having matching male and female tissues), and KEGG pathways and GO enrichments.

Overall, this is an exciting development in the study of echinoids and echinoderms. This manuscript can be improved by providing the reader with more details. Given the exciting prospect of being able to study M. globulus settlement and metamorphosis in detail, the authors may consider providing more information about this part of the life cycle.

Author response:

Reviewer #1 (Public review):

(1) The efficiency of the CRISPR/Cas9 knockout is illustrated qualitatively, but no sample size or penetrance value is reported, making it difficult for readers to judge how robust or reproducible this result is.

We agree that quantitative documentation of the knockout experiments is needed. In the revised manuscript we will report the number of injected embryos, the number of independent experiments, and the penetrance of the albino phenotype obtained with the PKS guide RNAs, so that readers can judge the robustness and reproducibility of gene editing in M. globulus.

(2) The gene annotation is reported to have complete PFAM domain coverage for only 75% of predicted genes, but no independent completeness metric (such as BUSCO scored against the annotated gene set rather than the assembly) is provided, leaving open whether the remaining genes are genuinely novel, partial models, or annotation artefacts.

We have now scored BUSCO (metazoa_odb10, n = 954) directly against the annotated gene sets rather than the assemblies. The annotation of the blue male genome is 96.9% complete (S: 89.7%, D: 7.2%; F: 1.2%, M: 1.9%), and the annotation of the red female genome is 97.6% complete (S: 97.0%, D: 0.6%; F: 0.8%, M: 1.6%). These values indicate that the predicted gene sets show a high degree of completeness and that the genes lacking full PFAM domain coverage are not simply the product of fragmented or artefactual gene models. These metrics will be added to the revised manuscript.

(3) Finally, at the time of review the NCBI BioProject accession cited for the genome and sequencing data (PRJNA1477966) could not be located, and it is not clear from the text whether this accession, once available, will include the gene annotation and RNA-seq datasets in addition to the raw genomic sequencing reads.

We apologise for not commissioning the release of these records, the BioProject was still being processed by NCBI at the time of review. We confirm that the accession will include the gene annotations and the RNA-seq datasets in addition to the raw genomic sequencing reads, and we will verify that all records are publicly accessible before submitting our revision. We will also state explicitly in the Data Availability section which datasets are deposited under this accession.

Reviewer #2 (Public review):

(1) Genetic Background and Aquarium Trade Populations: A central argument of the manuscript is that M. globulus is attractive as a laboratory model because it is widely cultured in the aquarium trade and may exhibit reduced genetic variability due to captive propagation. [...] The manuscript does not provide sufficient information to evaluate these claims: how genetically representative are the sequenced individuals relative to natural populations; what is known about the provenance and breeding history of the aquarium trade stocks; are these animals derived from a small number of founder populations; is there evidence for substantial inbreeding or genetic bottlenecks within commercial brood stocks; and how similar are commercially available animals from different vendors and geographic sources?

We thank the reviewer for raising this important point, which we have investigated directly. The two sequenced colour morphs have distinct provenances: the red individual derives from a line bred in captivity in North America, whereas the blue individual is wild-caught from the Indo-Pacific. We therefore expected the captive-bred red animal to show reduced polymorphism relative to the wild blue animal. We will thoroughly check differences of polymorphism between and across individuals from either population and discuss the results.

(2) Presentation and Interpretation of HCR and Phalloidin Data: [...] The HCR images show detectable signal, but the expression domains are only minimally documented [...] Similarly, the phalloidin-labeled images provide limited anatomical information because the larvae are largely not labeled. I recommend: (1) adding labels identifying relevant embryonic regions and structures; (2) including arrows or overlays indicating key expression domains; (3) providing higher-magnification insets of relevant regions; (4) including selected optical sections rather than relying exclusively on 3D projections; (5) identifying known larval muscle groups in the phalloidin images; (6) improving image contrast and figure annotation where possible.

We agree that the HCR and phalloidin panels should stand on their own for readers who are not sea urchin specialists. The revised manuscript will implement the reviewer's suggestions and provide more detailed labelling of embryonic territories and high-magnificant insets.

Reviewer #3 (Public review):

First, the authors should provide a thorough description of the methods used to cultivate and maintain M. globulus. This should include further details about the closed aquarium system; a schematic of the system would be insightful. Basic details about husbandry are needed, including (i) stocking densities of adults, embryos/larvae, postlarvae/juveniles; (ii) frequencies of level and water changes/top-ups; (iii) feeding regime at all phases of the life cycle.

We agree that full documentation of the culture system is essential for uptake of the model, and we will expand the Methods accordingly. A construction schematic of the closed aquarium system, produced for us by Aquarium Connections (London, UK), is provided in Author response image 1 and will be included in the revised manuscript as a supplementary figure. The system is a three-tier rack (2000 x 1800 x 700 mm) comprising a brood-stock holding tank with egg-crate divisions, a row of settlement tanks, and a lower sump level with a UKS 200 protein skimmer, XHO algae lighting for live-feed culture, and a reverse-osmosis top-up reservoir, controlled via timer and switch boxes.

The revised Methods will include the following husbandry parameters. Adults are stocked at 20 M. globulus per 300 L tank and fed kombu seaweed once per week. Embryos are stocked at 1 embryo per 4 mL, which corresponds to the density required at the larval stage; approximately 20% of larvae are lost by the time competency is reached. Once feeding begins, larval cultures are cleaned every three days, with water topped up at the same time. The day-by-day larval feeding and rearing schedule, from fertilisation through metamorphosis and the transition to the juvenile diet, is summarised in Author response table 1 and will be included in the revised Methods. We agree that this documentation will provide the foundation for future improvements, including shortening time to competence and maturity and standardising settlement.

Author response table 1.

Larval feeding and rearing schedule for M. globulus.

Author response image 1.

Construction plan of the closed M. globulus culture system (Aquarium Connections, drawing GC-001, rev. P1; scale 1:10 at A3). The three-tier rack (2000 x 1800 x 700 mm) houses the brood-stock holding tank with egg-crate divisions (top), settlement tanks (middle), and sump level with UKS 200 protein skimmer, XHO algae lighting, and RO top-up reservoir (bottom).

Second, the description of the procurement and analysis of mRNA is brief, unreferenced and reads as protocols used for an established model species (e.g. what is PFA in this case - the concentration of paraformaldehyde and the buffer can vary markedly between organisms and life stages). Even the RNA extraction protocols can vary between and within species. [...] The HCR analysis, which is also scantily described, is restricted to embryonic and larval stages. Given the emphasis on the capacity of the M. globulus system to analyse all phases of the life cycle, it would be good to know if HCR can be performed on settled postlarvae, juveniles and adult tissues.

We will substantially expand the Methods to give a complete, referenced account of fixation (including the paraformaldehyde concentration and buffer used at each stage), RNA extraction, and the HCR protocol as applied to M. globulus, rather than assuming familiarity with protocols from established models. We will also address the applicability of HCR beyond embryonic and larval stages in the revision. We agree that demonstrating in situ methods in post-settlement stages would reinforce the central premise of the model, and we will report our experience with postlarval, juvenile and adult material in the revised manuscript.

Third, the authors should consider dividing Figure 2, which consists of confocal images of normal development, HCR results and CRISPR/Cas9 knockout results, into three separate figures that explore these studies separately. A figure on normal development could, for instance, include documentation of metamorphosis, with a suite of images of postlarval stages. A figure documenting HCR could be expanded to include more stages, higher magnification images and other genes. A figure on the Cas9 knockdown of a PKS gene can provide details on the normal expression of this gene.

We agree that Figure 2 is currently overloaded. In the revision we plan to split it into separate figures: one devoted to confocal documentation of normal development. We intend to further include one (or more) juvenile stage presenting the HCR expression data together with the CRISPR/Cas9 knockout results, with the improved annotation described in our response to Reviewer #2.

Fourth, there should be consideration of providing more characterisation about the protein-coding genes comprising the chromosomal region (Chr. 4) that has marked differences between sexes. This could go beyond Supplementary Table 4 and Supplementary Figure 5B, and include analysis of expression in adult tissues (this would be enhanced by having matching male and female tissues), and KEGG pathways and GO enrichments.

We agree that the sex-differentiated region on chromosome 4 deserves fuller characterisation. In the revised manuscript we will extend the analysis of the protein-coding genes in this region beyond Supplementary Table 4 and Supplementary Figure 5B, including functional characterisation (GO and KEGG enrichment) and, where material permits, analysis of expression in adult tissues.