Connexins evolved after early chordates lost innexin diversity

  1. Georg Welzel
  2. Stefan Schuster

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    Evaluation Summary:

    This paper addresses the question of why invertebrates use innexins and vertebrates connexins to form gap junctions. The authors survey genomic data across animal diversity to search for innexins and connexins and analyse the distribution of glycosylation sites in the extracellular loops of these proteins. The reported data support the hypothesis that connexins replaced innexins in chordate gap junctions due to an evolutionary bottle neck. Overall, the data were properly analyzed, but could be improved with respect to the sequence data for some phyla and the discussion from the results obtained.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Gap junction channels are formed by two unrelated protein families. Non-chordates use the primordial innexins, while chordates use connexins that superseded the gap junction function of innexins. Chordates retained innexin-homologs, but N-glycosylation prevents them from forming gap junctions. It is puzzling why chordates seem to exclusively use the new gap junction protein and why no chordates should exist that use non-glycosylated innexins to form gap junctions. Here, we identified glycosylation sites of 2388 innexins from 174 non-chordate and 276 chordate species. Among all chordates, we found not a single innexin without glycosylation sites. Surprisingly, the glycosylation motif is also widespread among non-chordate innexins indicating that glycosylated innexins are not a novelty of chordates. In addition, we discovered a loss of innexin diversity during early chordate evolution. Most importantly, lancelets, which lack connexins, exclusively possess only one highly conserved innexin with one glycosylation site. A bottleneck effect might thus explain why connexins have become the only protein used to form chordate gap junctions.

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  1. Version published to 10.7554/elife.74422 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Welzel and Schuster propose an interesting hypothesis that connexins replaced innexins in chordate gap junctions due to an evolutionary bottle neck. The majority of the animal phyla possess multiple innexins, and some of them form gap junctions while others do not. Chordates have only three innexins which are unable to form gap junctions due to glycosylation of extracellular loops; chordate gap junctions are built of connexins. The authors analysed innexin sequences from multiple chordate and non-chordate phyla and discovered that non-chordates possess both types of innexins: with and without N-glycosylation sites in their extracellular loops. Because in chordates there are only glycosylated innexins, the authors conclude that non-glycosylated innexins were lost in the last common ancestor of chordates (bottleneck effect). While in Lancelets there are no connexins, they are present in Tunicates evidencing that they evolved in the last common ancestor of Tunicates and Vertebrates.

    Strengths

    The strength of this work is that the authors analysed over 2000 innexins from multiple animal groups: seven non-chordate phyla and nine groups of chordates; both non-bilaterian and bilaterian phyla were included. This comprehensive dataset allowed the authors to propose a general scenario of gap junction evolution. In my opinion, the bioinformatics analysis reported in this work convincingly support the bottleneck mechanism in the innexin evolution.

    Weaknesses

    The weaknesses of the work are rather minor and do not affect the main conclusions: (1) There is no experimental proof that the glycosylation occurs on the same motives in all the animal phyla. However, such experiments would be quite challenging therefore bioinformatics prediction is an acceptable approach. (2) There is no experimental proof that the glycosylated innexins of Lancelets and Tunicates don't form a gap junction. In some animals glycosylated innexins still do form gap junctions therefore it is possible that it is the case in Lancelets and Tunicates too, especially considering that the glycosylation sites are not conserved between Vertebrates, Tunicates and Lancelets. In this case it would mean that after the loss of multiple innexins in the last common ancestor of chordates, innexins lost the ability to form gap junctions only in vertebrates.

    We now discus this scenario in more detail (page 8). It is correct that the glycosylation sites are conserved across the vertebrates but not also between them, the tunicates and lancelets. However, the alignment of the four lancelet innexins – that we now have additionally included to Figure 2 – demonstrates that the glycosylation sites are highly conserved within lancelets. We fully agree that there is no experimental proof that the lancelet innexins do not form gap junctions and include this possibility.

    (3) It is not clear if the authors aimed to use only genomic data or both genomic and transcriptomic. While it is stated that "The taxonomic groups that we have analyzed in this study were constrained by the availability of publicly available genomic data", the majority of datasets available on the Neurobase (used in this study) are ctenophore transcriptomes; the only ctenophore genome dataset is from Pleurobrachia bachei. Currently there are two other ctenophore genomes available: from Mnemiopsis leidyi (Ryan JF et al, 2013) and Hormiphora calfornensis (Schultz D et al, 2021). Additionally, genomic data are available for more cnidarian species too (e.g. sea anemones Nematostella vectensis, Exaiptasia pallida, Actinia tenebrosa and multiple corals (Shinzato C et al, 2020)).

    Thank you for pointing this out. We added the missing information about the transcriptomic data to the materials and methods section. In addition, we also added the innexin sequences of 14 additional species to our analysis (3 ctenophores and 11 cnidarians).

    (4) Line 210: “innexins were recruited as gap junction proteins in the common cnidarian/bilaterian ancestor” - gap junctions have been reported in ctenophores as well (Satterlie RA & Case JF, 1978) therefore it probably happened much earlier (in the last common ancestor of animals).

    We agree, thank you! We clarified this. (page 8, line 204).

    Reviewer #2 (Public Review):

    1. Understanding exactly the situation in chordates and non-chordate deuterostomes is key to accurately reconstructing the evolutionary steps at the base of chordates. The authors should increase their sampling in these important groups and include hemichordates and other xenambulacrarians.

    We absolutely agree and have increased the sampling at the base of chordates including genomic as well as transcriptomic data of xenacoelomorphs, echinoderms and hemichordates into our analysis. We feel that it really was worth the effort: We identified innexins in 4 xenacoelomorphs and in 4 echinoderms (where previously no innexins were known). We also found innexin-like fragments in hemichordate transcriptomes suggesting that also hemichordates have innexins. We did not find connexins in these taxa (see Fig. 3).

    In Fig. 2. the alignments could include the non-vertebrate chordates (tunicate, lancelet) and lampreys to show whether the NGS sites are conserved in these taxa.

    Yes, this is a good point! We include innexin alignments of tunicates, lancelets and lampreys in Fig. 2.

    Tunicates have both innexins and connexins, does the NGS in innexin align to that of vertebrates?

    The NGS in tunicates are highly variable and do not align to that of vertebrates. We assume that this is due to the extremely fast evolution of the tunicate genomes and the high amino acid evolutionary rates in tunicates. We have discussed the conservation of NGS in tunicates (page 6, line 144).

    Please also show the situation with hemichordates in Fig 3.

    Yes, see above.

    1. The authors should discuss the genomic patterns also in light of the ultrastructural evidence from the literature. For example, their data suggest that cephalochordates lack gap junctions.

    "The most important finding is that the sequence of the only innexin of lancelets, which do not yet express connexins (Mikalsen et al., 2021; Slivko-Koltchik et al., 2019) (Figure 3D), contains a NGS in its extracellular loop 1. This suggests that the most basal chordates not only had a limited number of innexins but might also not be able to form functional gap junctions"

    Does this mean that lancelets have no gap junctions? The authors in particular should check and discuss these studies:

    Tissue and Cell Volume 19, Issue 3, 1987, Pages 399-411 Cell junctions in amphioxus (Cephalochordata): A thin section and freeze-fracture study https://doi.org/10.1016/0040-8166(87)90035-8

    This study finds no gap junctions in amphioxus epidermis, alimentary tract and notochord.

    Primary Sensory Cells in the Skin of Amphioxus (Branchiostoma lanceolatum (P)) Erik Baatrup, 1981 https://doi.org/10.1111/j.1463-6395.1981.tb00624.x

    In particular: "This agrees with the description (Baskin 1975) of the epidermal junctional complex of Branchiostoma californiense, but in addition this author found a membrane apposition resembling a gap junction. This was not observed in the present investigation of Branchiostoma lanceolatum.""

    but some authors described gap junction like structures https://europepmc.org/article/med/2628486

    Gap junctions are common in tunicates, this should also be mentioned:

    Georges, 1979 D. Georges, Gap and tight junctions in tunicates. Study in conventional and freeze-fracture techniques Tissue & Cell, 11 (1979), pp. 781-792

    In echinoderms, there are gap junctions but no connexins BMC Evol Biol. 2019 Feb 26;19(Suppl 1):46. doi: 10.1186/s12862-019-1369-4. Are there gap junctions without connexins or pannexins?

    Georgy A Slivko-Koltchik 1 , Victor P Kuznetsov 1 , Yuri V Panchin 2 3 PMID: 30813901 PMCID: PMC6391747 DOI: 10.1186/s12862-019-1369-4

    Thank you for pointing this out. We have discussed the question whether lancelets have gap junctions or not in the revised manuscript and added the suggested literature (page 8, line 190).

    Reviewer #3 (Public Review):

    The gap junction-forming proteins, vertebrate connexins and invertebrate innexins, are two distinct protein families with very similar structures and functions. In the process of evolution, innexins first arose in invertebrates, followed by connexins in vertebrates.

    The authors focused on the extracellular glycosylation site in innexins, that inhibit channel coupling between two cells, and analyzed available innexin sequences using the genomic database and reported sequences.

    The results showed, as phylogenetic evolution progresses, innexins lose their diversity and converge only on innexins that undergo glycosylation. And connexins without glycosylation sites arose as new gap junction-forming proteins. The authors proposed a new evolutionary scenario in which the switching of gap junction protein from invertebrate innexins to vertebrate connexins is due to the loss of diversity (especially glycosylation) of innexins.

    Strengths:

    This study, which focuses on the molecular evolution involved in the biologically important mechanism of gap junctions, is significant, and will influence many future studies. Overall, the data were properly analyzed, and the visible diagrams have been created based on a vast amount of analysis.

    Weaknesses:

    1. The authors discussed the decrease or appearance of specific genes based on the results obtained from comprehensive sequence analysis. However, in order to discuss the number of specific genes in each animal species, especially to prove that a particular gene does not exist, the quantity and quality of the genome database greatly affect the results. It is unlikely that no gap junction proteins present at all in Echinoderms. For animal phyla for which accurate sequence data are scarce, an additional search that includes TSA will yield better results.

    Thank you very much for pointing this out. As suggested by you as well as by reviewer 1, we have additionally searched for innexins and connexins in the non-chordate deuterostomes (xenacoelomorphs, echinoderms, hemichordates) as well as lancelets, tunicates and lampreys by including the NCBI TSA databases. By doing this, we additionally found innexins of two lancelets, four xenacoelomorphs and four echinoderms.

    1. The authors proposed a scenario in which connexins emerged due to the loss of gap junction forming ability of innexins during evolution. However, this study focused only on the presence or absence of glycosylation modifications and did not consider the number of proteins in the innexin and connexin families per each animal species. Normally, gap junction-forming proteins have multiple family proteins in each animal species, and these proteins are combined to regulate channel function. The authors' scenario does not explain the small number of variety of innexin and connexin family proteins found in each phylum of Echinoderms and lancelets, and this needs to be discussed.

    Thank you for pointing out this key aspect! Above all, it is the loss of functional diversity of putative gap junctions that is cause by having only one type of innexin. We therefore already prepared this crucial point in the introduction and before introducing the bottleneck idea. It is a very important idea to make clear how a small number of innexins limits the number of functionally specialized combinations of heterotypic/heteromeric gap junction channels (page 2 and 8). It is also a good idea to directly display the mean number of connexins and innexins per each animal species in Figure 3B. This also should help to emphasize our main point: the decrease of innexin diversity and the increase of connexin diversity during early chordate evolution.

  3. eLife

    Evaluation Summary:

    This paper addresses the question of why invertebrates use innexins and vertebrates connexins to form gap junctions. The authors survey genomic data across animal diversity to search for innexins and connexins and analyse the distribution of glycosylation sites in the extracellular loops of these proteins. The reported data support the hypothesis that connexins replaced innexins in chordate gap junctions due to an evolutionary bottle neck. Overall, the data were properly analyzed, but could be improved with respect to the sequence data for some phyla and the discussion from the results obtained.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Welzel and Schuster propose an interesting hypothesis that connexins replaced innexins in chordate gap junctions due to an evolutionary bottle neck. The majority of the animal phyla possess multiple innexins, and some of them form gap junctions while others do not. Chordates have only three innexins which are unable to form gap junctions due to glycosylation of extracellular loops; chordate gap junctions are built of connexins. The authors analysed innexin sequences from multiple chordate and non-chordate phyla and discovered that non-chordates possess both types of innexins: with and without N-glycosylation sites in their extracellular loops. Because in chordates there are only glycosylated innexins, the authors conclude that non-glycosylated innexins were lost in the last common ancestor of chordates (bottleneck effect). While in Lancelets there are no connexins, they are present in Tunicates evidencing that they evolved in the last common ancestor of Tunicates and Vertebrates.

    Strengths
    The strength of this work is that the authors analysed over 2000 innexins from multiple animal groups: seven non-chordate phyla and nine groups of chordates; both non-bilaterian and bilaterian phyla were included. This comprehensive dataset allowed the authors to propose a general scenario of gap junction evolution. In my opinion, the bioinformatics analysis reported in this work convincingly support the bottleneck mechanism in the innexin evolution.

    Weaknesses
    The weaknesses of the work are rather minor and do not affect the main conclusions:
    (1) There is no experimental proof that the glycosylation occurs on the same motives in all the animal phyla. However, such experiments would be quite challenging therefore bioinformatics prediction is an acceptable approach.
    (2) There is no experimental proof that the glycosylated innexins of Lancelets and Tunicates don't form a gap junction. In some animals glycosylated innexins still do form gap junctions therefore it is possible that it is the case in Lancelets and Tunicates too, especially considering that the glycosylation sites are not conserved between Vertebrates, Tunicates and Lancelets. In this case it would mean that after the loss of multiple innexins in the last common ancestor of chordates, innexins lost the ability to form gap junctions only in vertebrates.
    (3) It is not clear if the authors aimed to use only genomic data or both genomic and transcriptomic. While it is stated that "The taxonomic groups that we have analyzed in this study were constrained by the availability of publicly available genomic data", the majority of datasets available on the Neurobase (used in this study) are ctenophore transcriptomes; the only ctenophore genome dataset is from Pleurobrachia bachei. Currently there are two other ctenophore genomes available: from Mnemiopsis leidyi (Ryan JF et al, 2013) and Hormiphora calfornensis (Schultz D et al, 2021). Additionally, genomic data are available for more cnidarian species too (e.g. sea anemones Nematostella vectensis, Exaiptasia pallida, Actinia tenebrosa and multiple corals (Shinzato C et al, 2020)).
    (4) Line 210: «innexins were recruited as gap junction proteins in the common cnidarian/bilaterian ancestor» - gap junctions have been reported in ctenophores as well (Satterlie RA & Case JF, 1978) therefore it probably happened much earlier (in the last common ancestor of animals).

  5. eLife

    Reviewer #2 (Public Review):

    1. Understanding exactly the situation in chordates and non-chordate deuterostomes is key to accurately reconstructing the evolutionary steps at the base of chordates. The authors should increase their sampling in these important groups and include hemichordates and other xenambulacrarians.
      In Fig. 2. the alignments could include the non-vertebrate chordates (tunicate, lancelet) and lampreys to show whether the NGS sites are conserved in these taxa.
      Tunicates have both innexins and connexins, does the NGS in innexin align to that of vertebrates?
      Please also show the situation with hemichordates in Fig 3.

    2. The authors should discuss the genomic patterns also in light of the ultrastructural evidence from the literature. For example, their data suggest that cephalochordates lack gap junctions.

    "The most important finding is that the sequence of the only innexin of lancelets, which do not yet express connexins
    (Mikalsen et al., 2021; Slivko-Koltchik et al., 2019) (Figure 3D), contains a NGS in its extracellular loop 1. This suggests that the most basal chordates not only had a limited number of innexins but might also not be able to form functional gap junctions"

    Does this mean that lancelets have no gap junctions? The authors in particular should check and discuss these studies:

    Tissue and Cell Volume 19, Issue 3, 1987, Pages 399-411
    Cell junctions in amphioxus (Cephalochordata): A thin section and freeze-fracture study
    https://doi.org/10.1016/0040-8166(87)90035-8

    This study finds no gap junctions in amphioxus epidermis, alimentary tract and notochord.

    Primary Sensory Cells in the Skin of Amphioxus (Branchiostoma lanceolatum (P))
    Erik Baatrup, 1981 https://doi.org/10.1111/j.1463-6395.1981.tb00624.x

    In particular:
    "This agrees with the description (Baskin 1975) of the epidermal junctional complex of Branchiostoma californiense, but in addition this author found a membrane apposition resembling a gap junction. This was not observed in the present investigation of Branchiostoma lanceolatum.""

    but some authors described gap junction like structures
    https://europepmc.org/article/med/2628486

    Gap junctions are common in tunicates, this should also be mentioned:

    Georges, 1979 D. Georges
    Gap and tight junctions in tunicates. Study in conventional and freeze-fracture techniques
    Tissue & Cell, 11 (1979), pp. 781-792

    In echinoderms, there are gap junctions but no connexins
    BMC Evol Biol. 2019 Feb 26;19(Suppl 1):46. doi: 10.1186/s12862-019-1369-4.
    Are there gap junctions without connexins or pannexins?

    Georgy A Slivko-Koltchik 1 , Victor P Kuznetsov 1 , Yuri V Panchin 2 3
    PMID: 30813901 PMCID: PMC6391747 DOI: 10.1186/s12862-019-1369-4

  6. eLife

    Reviewer #3 (Public Review):

    The gap junction-forming proteins, vertebrate connexins and invertebrate innexins, are two distinct protein families with very similar structures and functions. In the process of evolution, innexins first arose in invertebrates, followed by connexins in vertebrates.

    The authors focused on the extracellular glycosylation site in innexins,　that inhibit channel coupling between two cells, and analyzed available innexin sequences using the genomic database and reported sequences.
    The results showed, as phylogenetic evolution progresses, innexins lose their diversity and converge only on innexins that undergo glycosylation. And connexins without glycosylation sites arose as new gap junction-forming proteins. The authors proposed a new evolutionary scenario in which the switching of gap junction protein from invertebrate innexins to vertebrate connexins is due to the loss of diversity (especially glycosylation) of innexins.

    Strengths:
    This study, which focuses on the molecular evolution involved in the biologically important mechanism of gap junctions, is significant, and will influence many future studies. Overall, the data were properly analyzed, and the visible diagrams have been created based on a vast amount of analysis.

    Weaknesses:

    1. The authors discussed the decrease or appearance of specific genes based on the results obtained from comprehensive sequence analysis. However, in order to discuss the number of specific genes in each animal species, especially to prove that a particular gene does not exist, the quantity and quality of the genome database greatly affect the results. It is unlikely that no gap junction proteins present at all in Echinoderms. For animal phyla for which accurate sequence data are scarce, an additional search that includes TSA will yield better results.

    2. The authors proposed a scenario in which connexins emerged due to the loss of gap junction forming ability of innexins during evolution. However, this study focused only on the presence or absence of glycosylation modifications and did not consider the number of proteins in the innexin and connexin families per each animal species. Normally, gap junction-forming proteins have multiple family proteins in each animal species, and these proteins are combined to regulate channel function. The authors' scenario does not explain the small number of variety of innexin and connexin family proteins found in each phylum of Echinoderms and lancelets, and this needs to be discussed.

  7. Version published to 10.1101/2021.10.08.463644v1 on bioRxiv