Evolutionary phenome-genome analysis of cranial suture closure in mammals

  1. Borja Esteve-Altava
  2. Fabio Barteri
  3. Xavier Farré
  4. Gerard Muntané
  5. Juan Francisco Pastor
  6. Arcadi Navarro

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Abstract

Cranial sutures are growth and stress diffusion sites that connect the bones protecting the brain. The closure of cranial suture is a key feature of mammalian late development and evolution, which can also lead to head malformations when it occurs prematurely (craniosynostosis). To unveil the phenotypic and genetic causes of suture closure in evolution, we examined 48 mammalian species searching for (i) causal links between suture patency, brain size, and diet using phylogenetic path analysis; and (ii) instances of genome-phenome convergence amino acid substitutions. Here we show that brain size and the anteroposterior order of ossification of the skull are the two main causes of sutures patency in evolution. We also identified three novel candidate genes for suture closure in evolution (HRNR, KIAA1549 , and TTN ), which have never been reported in clinical studies of craniosynostosis. Our results suggest that different genetic pathways underlie cranial suture closure in evolution and disease.

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  1. eLife

    ##Author Response

    ###Reviewer #1:

    This study was designed to determine whether there is a relationship among cranial suture closure patterns, the molecular causes for suture patency/closure, and phylogeny. The authors use correlative data to test causal hypotheses related to brain size, suture closure patterns, and diet and search for the genetic underpinnings of the relationships they identify using reference genomes. There are many ideas put forward and methods used that are not clearly explained in the body of the work or in the supplementary material. This made it difficult to provide a clear evaluation of the work. Even checking original sources on which they base their approach, I found some disconnect between original sources and ideas laid out here. I see some interesting ideas in the study but a lack of solid reasoning behind the hypotheses proposed, confusion about the data and/or ideas summarized from the literature (the confusion could be on my part, but it rests with the authors to explain this more fully), and lack of detail regarding methods used to support their conclusions.

    We take good note of this confusion and we will explain everything in more detail in a revised version of the manuscript.

    1. The entire study rests on the authors scoring of sutures as patent or closed but no information is given other than a suture was considered closed if it was not visible ( 'obliterated"), and a suture was considered open if visible. These are problematic definitions for distinguishing patent from closed sutures if we accept the authors' definition of sutures as growth and stress diffusion sites. A suture can be visible but still be "closed" as evidenced by bony connections or bridges linking the bones that border the suture. In the case of bridging, the suture would be visible, so would be scored as "open" according to the authors' criterion, but functionally, the suture is closed.

    Visual examination of sutures (e.g., from photos or in situ) is a common procedure in macroevolutionary studies of suture patency, where raw data is not always available for histological inspection (e.g., invasive procedures or CT are not permitted). In this regard, we follow previous literature. We would like to note that only photographic materials were available for most specimens during this project, because of the current exceptional circumstances (museums lockdown).

    Also, in some mammals (e.g., the laboratory mouse) most cranial sutures do not close in typically developing individuals.

    In this study we used specimens hosted in museum collections, which come from the wild or zoos. We did not use data from laboratory animals grown in controlled environments, which may indeed affect their suture patency (e.g., by feeding on pellets).

    1. Age estimates are not provided for the specimens used in analysis. In many mammalian species, suture closure occurs in a somewhat predictable fashion - this, coupled with tooth formation/eruption patterns is one of the ways that forensic scientists aged skeletal remains prior to the advent of modern technologies. The order of suture closure is not necessarily similar across vertebrates, or even across mammals. This means that, without known or estimated ages for each skull included in analysis, age becomes an unrecognized source of variation that will affect analytical outcome.

    Unfortunately, the exact age for museum specimens is often not available. For this reason, we focused on adult specimens, where suture patency tends to remain constant. We also excluded individuals with signs of senescence. To accommodate age and other source of intraspecific variation in adults, we collected information for as many individuals as possible, often more than 10 and sometimes up to 100. Thus, we coded suture patency as a frequency rather than as open/closed for each species.

    We only dichotomized suture patency as open/closed for the second part of the study. Here we used a sensible threshold to avoid ambiguity and be conservative. As a result, species with frequency of suture patency between 75% and 25% were excluded. This also means that if only 4 individuals were examined (small sample size was unavoidable for some rare species) and at least one showed a discrepancy, that species was excluded from the analysis. However, because suture patency is a very conserved trait, only a few taxa had to be excluded at the end.

    In any case, we will emphasize more this fact in the revised version.

    1. The authors' impact statement: "brain growth and skull ossification sequence cause suture closure in mammals evolution without common genetic factors causing premature suture closure diseases in humans" is hard to digest as brain growth is not considered by the authors but instead brain size. From a developmental perspective, brain size or even some form of the encephalization quotient (EQ) is not what is commonly proposed to drive suture closure/patency (or degree of patency). Instead it is the dynamics of brain growth that is proposed as a stimulus for the initiation of mineralization of cranial bones. As bones increase in size, new bone is added at the leading edge of opposing bones that line the suture, while the stem cells in the center of the suture remain to add to the mesenchymal cell population of the suture, keeping the suture patent. In short, the dynamics of brain growth (including any signaling emanating from the brain, dura, bones, or even the suture itself) contributes to suture patency. Because sutures tend to close later in life (after childhood in humans), normal suture closure appears to be associated with the termination of brain growth. Making the jump in their study from estimates of EQ (in some way estimated here) to dynamics of brain growth as a cause requires several steps and knowledge on timing and rate of growth that is not considered by the authors.

    We agree with the reviewer. A developmentally focused study on suture formation and closure dynamics must consider brain growth. However, this information is not available for most species selected for this study. Note that species selection depended on the availability of referenced genomes and multiple sequence alignments (some of which are rare, endangered species). Because we were comparing macroevolutionary dynamics in adults we decided to use brain size as a feasible proxy for brain influence (either due to growth or signalling). We aim to fill this gap in future research projects. In the meantime, we will revise the wording of the article to make sure that there are no misleading statements about brain growth influence.

    1. The authors assume a suture closure pattern across the skull that starts at the anterior (rostrally) and move posteriorly (caudally) and builds this into their model. This seems to be based on a work by Koyabu et al. (2014), but that study is about the appearance of ossification centers for bones (not suture formation or closure) and the study actually clumps the frontal and parietal into the same group in their final analysis so why this supports and anterior to posterior direction of suture closure is not clear.

    Note that we did not “assume” any closure pattern; we interpreted the published evidence on how the skull ossifies in mammals to make a plausible hypothesis. We also tested other 11 plausible hypotheses. It could have happened that such hypothesis was worse than the others, but we found that the best supported hypothesis includes an anterior-posterior relation of suture closure. We will try to explain the construction of our model and hypothesis testing better in the revised version.

    1. The authors conclusion: (Lines 289-292 does not follow from their analyses.) Brain growth was not analyzed. I am uncertain what they mean by suture self-regulation as I don't think their detection of genetic variants in common across a diverse set of species means that those are controlling suture patency/closure.

    The proposed idea of suture self-regulation refers to the fact that one suture closure may affect another suture closure (as theoretical models previously suggested), and it is not necessarily related to the genetic variants identified here. As explained before, we will revise any reference to brain growth.

    ###Reviewer #2:

    -Authors tested 4 hypotheses (page 5, lines 78-84), but rejected or questioned them later on (which is a fair approach to be realistic and point out possible weaknesses or methodological limitations, nevertheless, I find there are more questions or suggestions rather than actual answers).

    We have tried to offer an open and clear set of hypotheses, tested them with the available data, and discussed the results fairly. As it is often the case in science, research may bring more questions than answers; we do not see this as a weakness. Our answers are also contextualized within the limitations that we described in the methods. We believe this is the correct way of doing science: even if this forces us to reject all our hypotheses, negative results are also results. Since our object of study is not very well known, we hope this study can fuel more research.

    -Lots of repeating text

    -Frequent missing references for major statements, unclear formulations

    We will double-check our manuscript. However, the reviewer offers no details about what is repeated or missing.

    -Few contradicting or unclear information, for instance, "high conservation..enabled us to categorize phenotype as either open or closed" / "suture patency ranging from 0-1, only above 75% and below 25% was counted as open or closed" / authors involved species were >2 samples were available but excluded any ambiguous case (small number of samples per species?)

    As explained before, thresholding at 25/75 % was used to binarize species as having a suture open or closed. This binarization is only used for the convergent amino acid substation analysis. We excluded ambiguous cases (i.e., a suture half closed) prior to data collection. We will explain it better in the revised version to avoid confusion.

    -"Phylogenetic path analysis showed almost no effect of diet on the brain size; low to medium (what does that mean then?) effect of brain on suture closure and medium to high effect of 1 suture affecting the other sutures in AP direction" (in many species this is described-the timeline of suture closure)

    Not sure about what the reviewer means; we will revise these sentences to make them clearer to readers.

    -I am not able to evaluate if the assessment of diet hardness as an equivalent to mechanical forces in the skull is correct and hope other reviewers will be able to do that-in fact, also to evaluate the phylogenetic path analysis performed in this manuscript. Authors took information on % of nektar/soft-plants and invertebrates/hard food (seeds etc) that given species consumes and multiplied by an index but not an actual modeling or assessment of the forces... To a laymen it looks like, for instance, cow chewing all day long relatively soft grass, building very strong muscles will at the end develop much more force/tension within the skull than an animal cracking one nut.

    As the reviewer correctly points out, chewing grass all day long is harder than cracking one nut (cracking nuts “all day long” would be another issue). In any case, we have weighed each food item compared to others (e.g., grass is weighed as twice as hard as meat) and there is consensus that feeding on seeds and scavenging is one of the most biomechanically demanding feeding strategies. In addition, we would like to note that we critically discussed the caveats of diet hardness as a proxy for the effect of feeding biomechanics on sutures, and we did not blindly assume this as a hard truth.

    -Lots of attention is given to the three identified genes with convergent amino acid substitution despite the fact that none of these genes have ever been related to any aspect of craniofacial biology, nor to the suture pathological conditions.

    We discussed the three genes that our analysis revealed. We cannot discuss genes for which we found no support. For these three genes, we offered plausible scenarios for how they could be associated to craniosynostosis; it is for future studies to explore these scenarios and validate experimentally or clinically these genes. The fact that they are not currently known as part of pathological conditions does not preclude that we need to discuss them in the manuscript. Every year, new genetic variants are discovered to be associated with craniosynostosis. The lack of correspondence between these genes and pathology is in fact one of the findings of this study: the few genes that show convergent mutations are not associated to pathology. We agree that absence of evidence is not evidence of absence. However, we also think that this is a result to be discussed in this manuscript and for the readers to ponder.

  2. eLife

    ###Reviewer #2:

    Authors' goal was to reveal phenotypic and genetic causes of suture closure in evolution. Authors formulated and tested several hypotheses to find out whether brain size, diet hardness, etc is a causal link to the presence of typically patent (open) or closed sutures in 48 mammalian species. Next, authors attempted to identify genes (And convergent AC substitutions) associated with these species-specific suture status, and relate them to the biological functions commonly associated with suture formation and/or mutation in pathological conditions such as craniosynostosis.

    While I think it is an interesting question or hypothesis to test (seems to be inspired by Abelson 2016 and similar studies) during the reading, several concerns arose (and even authors themselves pointed out several of them a few times). Overall, I do not find convincing evidence for the authors' statements. Very briefly, just few of my comments:

    -Authors tested 4 hypotheses (page 5, lines 78-84), but rejected or questioned them later on (which is a fair approach to be realistic and point out possible weaknesses or methodological limitations, nevertheless, I find there are more questions or suggestions rather than actual answers).

    -Lots of repeating text

    -Frequent missing references for major statements, unclear formulations

    -Few contradicting or unclear information, for instance, "high conservation..enabled us to categorize phenotype as either open or closed" / "suture patency ranging from 0-1, only above 75% and below 25% was counted as open or closed" / authors involved species were >2 samples were available but excluded any ambiguous case (small number of samples per species?)

    -"Phylogenetic path analysis showed almost no effect of diet on the brain size; low to medium (what does that mean then?) effect of brain on suture closure and medium to high effect of 1 suture affecting the other sutures in AP direction" (in many species this is described-the timeline of suture closure)

    -I am not able to evaluate if the assessment of diet hardness as an equivalent to mechanical forces in the skull is correct and hope other reviewers will be able to do that-in fact, also to evaluate the phylogenetic path analysis performed in this manuscript. Authors took information on % of nektar/soft-plants and invertebrates/hard food (seeds etc) that given species consumes and multiplied by an index but not an actual modeling or assessment of the forces... To a laymen it looks like, for instance, cow chewing all day long relatively soft grass, building very strong muscles will at the end develop much more force/tension within the skull than an animal cracking one nut.

    -Lots of attention is given to the three identified genes with convergent amino acid substitution despite the fact that none of these genes have ever been related to any aspect of craniofacial biology, nor to the suture pathological conditions.

  3. eLife

    ###Reviewer #1:

    This study was designed to determine whether there is a relationship among cranial suture closure patterns, the molecular causes for suture patency/closure, and phylogeny. The authors use correlative data to test causal hypotheses related to brain size, suture closure patterns, and diet and search for the genetic underpinnings of the relationships they identify using reference genomes. There are many ideas put forward and methods used that are not clearly explained in the body of the work or in the supplementary material. This made it difficult to provide a clear evaluation of the work. Even checking original sources on which they base their approach, I found some disconnect between original sources and ideas laid out here. I see some interesting ideas in the study but a lack of solid reasoning behind the hypotheses proposed, confusion about the data and/or ideas summarized from the literature (the confusion could be on my part, but it rests with the authors to explain this more fully), and lack of detail regarding methods used to support their conclusions.

    1. The entire study rests on the authors scoring of sutures as patent or closed but no information is given other than a suture was considered closed if it was not visible ( 'obliterated"), and a suture was considered open if visible. These are problematic definitions for distinguishing patent from closed sutures if we accept the authors' definition of sutures as growth and stress diffusion sites. A suture can be visible but still be "closed" as evidenced by bony connections or bridges linking the bones that border the suture. In the case of bridging, the suture would be visible, so would be scored as "open" according to the authors' criterion, but functionally, the suture is closed. Also, in some mammals (e.g., the laboratory mouse) most cranial sutures do not close in typically developing individuals.

    2. Age estimates are not provided for the specimens used in analysis. In many mammalian species, suture closure occurs in a somewhat predictable fashion - this, coupled with tooth formation/eruption patterns is one of the ways that forensic scientists aged skeletal remains prior to the advent of modern technologies. The order of suture closure is not necessarily similar across vertebrates, or even across mammals. This means that, without known or estimated ages for each skull included in analysis, age becomes an unrecognized source of variation that will affect analytical outcome.

    3. The authors' impact statement: "brain growth and skull ossification sequence cause suture closure in mammals evolution without common genetic factors causing premature suture closure diseases in humans" is hard to digest as brain growth is not considered by the authors but instead brain size. From a developmental perspective, brain size or even some form of the encephalization quotient (EQ) is not what is commonly proposed to drive suture closure/patency (or degree of patency). Instead it is the dynamics of brain growth that is proposed as a stimulus for the initiation of mineralization of cranial bones. As bones increase in size, new bone is added at the leading edge of opposing bones that line the suture, while the stem cells in the center of the suture remain to add to the mesenchymal cell population of the suture, keeping the suture patent. In short, the dynamics of brain growth (including any signaling emanating from the brain, dura, bones, or even the suture itself) contributes to suture patency. Because sutures tend to close later in life (after childhood in humans), normal suture closure appears to be associated with the termination of brain growth. Making the jump in their study from estimates of EQ (in some way estimated here) to dynamics of brain growth as a cause requires several steps and knowledge on timing and rate of growth that is not considered by the authors.

    4. The authors assume a suture closure pattern across the skull that starts at the anterior (rostrally) and move posteriorly (caudally) and builds this into their model. This seems to be based on a work by Koyabu et al. (2014), but that study is about the appearance of ossification centers for bones (not suture formation or closure) and the study actually clumps the frontal and parietal into the same group in their final analysis so why this supports and anterior to posterior direction of suture closure is not clear.

    5. The authors conclusion: (Lines 289-292 does not follow from their analyses.) Brain growth was not analyzed. I am uncertain what they mean by suture self-regulation as I don't think their detection of genetic variants in common across a diverse set of species means that those are controlling suture patency/closure.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

  5. Version published to 10.1101/2020.06.15.148130v1 on bioRxiv