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The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons

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Abstract

Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311–314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253–264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185–97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes, a property that may underlie the participation of these neurons in different adult coupling circuits.

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Notes

  1. We have adopted H. verbana (Hve) for the Hve-inx1-19 genes reported here, since these sequences are known to be derived from a specimen of this species. Previous reports of these genes were ascribed to the species H. medicinalis and are found in NCBI under Hm-inx1-12, but it is likely that the source was a mixture of the two species. The confusion between these two highly related species of the medicinal leech is discussed in Siddall et al. 2007. Throughout this paper, gene names will be lower case and italicized, and corresponding protein names will be capitalized, no italics. The species code Hve will be omitted except where interspecies comparisons require it.

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Acknowledgments

The costs of the work reported here were supported in part by U.S. National Science Foundation Grants IOB-0446346 and DBI-0852081.

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Authors and Affiliations

  1. Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, USA

    Brandon Kandarian, Jasmine Sethi, Allan Wu, Michael Baker, Neema Yazdani, Eunice Kym, Alejandro Sanchez, Lee Edsall, Terry Gaasterland & Eduardo Macagno

  2. Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY, 10029-6574, USA

    Brandon Kandarian

  3. The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA

    Jasmine Sethi & Alejandro Sanchez

  4. UCSF Pharmacy School, Box 0150, Room S-960, San Francisco, CA, 94143, USA

    Eunice Kym

  5. Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, CA, USA

    Lee Edsall & Terry Gaasterland

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  1. Brandon Kandarian
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Correspondence to Eduardo Macagno.

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Communicated by D. Weisblat

Brandon Kandarian, Jasmine Sethi, and Allan Wu contributed equally to this work and should be considered as the first coauthors.

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Supplemental Fig. 1

Sequence alignment of the 21 Hirudo verbana innexin proteins reported here. The program ClustalW was used to perform the multiple alignments of the innexin sequences, while Jalview (Waterhouse et al. 2009) was used to generate the image. The amino acids are colored according to the "Zappo" coloring scheme that denotes groups of residues with similar physicochemical properties. For example, the four transmembrane regions are predominantly pink, representing aliphatic or hydrophobic amino acids, while the two extracellular loops each display the highly conserved pair of cysteines in yellow. Other correspondences are: aromatic, orange; positive, blue; negative, red, hydrophilic, blue; and conformationally special, purple. The conservation score, quality score and consensus levels, indicated in three rows below the aligned sequences, were automatically calculated by the Jalview software, as described in the Jalview documentation (http://www.jalview.org/help.html) (PNG 474 kb)

Supplementary Fig. 2

Exons of the closely related Hve-INX9A and Hve-INX9B proteins, showing the seven predicted exons in different colors. Note that the exons are of identical lengths, with the splice site flanking amino acids highly conserved but not identical. Comparison of the predicted protein sequences of these two genes with the innexins of the leech Helobdella robusta indicates that both are orthologs of a single gene in that species, Hro-Inx9, suggesting that they arose from a recent duplication event (GIF 148 kb)

High-resolution image (TIFF 531 kb)

Supplementary Fig. 3

Phylogram comparing predicted innexin protein sequences from Hirudo verbana and Helobdella robusta, two distantly related species of leeches with extensive genomic sequence data. The accession numbers for the deposited sequence data are provided in Supplemental Table 1. With few exceptions, the alignment shows 1-to-1 homology between individual sequences from the two species. The exceptions (noted with brackets) comprise two proteins in each species that show 1-to-2 clustering. This is consistent with the genome of their common ancestor encoding 19 paralogs of the innexin genes, and with two of these in each species duplicating independently after speciation (GIF 111 kb)

High-resolution image (TIFF 235 kb)

Supplementary Fig. 4

Expression of inx13, a nonneuronal innexin, in stage E12 embryo whole mounts of Hirudo verbana. The ISH signal is highly localized to specific regions of the openings from the lumen of some body organs to the external world. The structures that are labeled are the nephridiopores (arrows in a and d), the gonopores (a); M and open arrow, male organ; F and open arrow, female organ) and the jaws (b), view into mouth; (c), tilted view, showing that expressing cells are located at the apical surfaces of the jaws. Bar at bottom left = 500 μm for a, 150 μm for b and c, and 50 μm for d (GIF 125 kb)

High-resolution image (TIFF 235 kb)

Supplementary Fig. 5

The expression of inx19 is much lower than the levels of the other innexins shown in Fig. 6b, but it shows a highly significant increase postembryonically (n = 3, p < 0.001) (GIF 28 kb)

High-resolution image (TIFF 101 kb)

Supplementary Table 1

Nomenclature, accession numbers and correspondence of the 21 Hirudo and 21 Helobdella innexin genes, as determined from sequence orthology analysis as described in the text. The analysis suggests that the ancestor of the Hirudo and Helobdella had 19 innexin genes, and that subsequent independent duplications raised that number to 21 in present day representative species (DOC 70 kb)

Supplementary Table 2

Accession numbers for the Capitella teleta innexin genes used in this report (DOC 60 kb)

Supplementary Table 3

Accession numbers for the Lottia gigantea innexin genes used in this report (DOC 51 kb)

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Kandarian, B., Sethi, J., Wu, A. et al. The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons. Dev Genes Evol 222, 29–44 (2012). https://doi.org/10.1007/s00427-011-0387-z

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