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Tail loss: investigating the Molgula

Tail loss: investigating the Molgula. C. Titus Brown Asst Professor, CSE / MMG Michigan State University. M.oculata. M.occulta. Molgula Clades – urodele (“tailed”) and anural (“tailless”) mix?!. Molgulids have many anural embryos – why?? (Ascidians generally: ~1% of 3,000 are tailless).

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Tail loss: investigating the Molgula

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  1. Tail loss: investigating the Molgula C. Titus Brown Asst Professor, CSE / MMG Michigan State University

  2. M.oculata M.occulta

  3. MolgulaClades – urodele (“tailed”) and anural (“tailless”) mix?! Molgulids have many anural embryos – why?? (Ascidians generally: ~1% of 3,000 are tailless)

  4. Station Biologique de Roscoff http://www.sb-roscoff.fr/ Seven species of Molgula described by H. Lacaze-Duthiers and his students (Swalla et al. found six of them)

  5. Two Species of Closely Related Ascidians and a Hybrid Larva Molgula oculata has a head, tail, and otolith - chordate embryo. M. occulta egg X M. oculata sperm has a head, short tail, and otolith. Molgula occulta lacks chordate features - head, tail, and otolith.

  6. Notochord Formation in Molgulids Molgula oculata notochord (40 cells, converged & extended) Molgula occulta no notochord (20 cells, not converged & extended) Hybrid notochord (20 cells, converged & extended) Swalla and Jeffery, 1996

  7. Brachyury Expression in M. oculata Hybrid M. occulta 8 + 2=10 8 + 2=10 8 + 2=10 10 x 2=20 10 x 2=20 10 x 2=20 Takada et al. (2002) Evol. & Dev. 4:3 205-211

  8. M. oculata M. occultaHybrids Larval muscle actin gene expression Absent in tailless species Tailed species gene is expressed in hybrids Jeffery WR,Swalla BJ et al. (1999) Mol. Biol. Evol. 16(5):646-654.

  9. In molgulid ascidians, manx and p68/bobcat are found in a gene complex and are required for tail development. manx p68 RNA helicase manx Swalla et al. (1999) Development 126:1643-1653

  10. Molgula questions • What happened to the downstream tail gene network in the tailless ascidian? • What are the genomic adaptations that made the Molgulidae particularly susceptible to tail loss? • How does tail loss actually work, functionally? • Heterochrony of metamorphosis?

  11. Approach • mRNA sequencing and quantitation from gastrula, neurula, and tailbud stages of M. oculata(tailed) and M. occulta(tailless). • Examine known molecular pathways. • Discovery-based approach => hypotheses. • Sequencing of hybrids at those same stages to identify likely cis changes. • Follow up with spatial analysis. • Eventually: Full integrative network analysis (genome + expression + perturbations)

  12. Allele specific expression • Detect haplotype-specific changes in gene expression as indicator of functional cis-variation.

  13. Short-read sequencing of mRNA • Deep, quantitative sampling of poly-A RNA population. • Can be used for expression analysis, transcriptome annotation (note, no reference!)

  14. Overall protocol

  15. Preliminary round of sequencing (Illumina 76 bp x 2, ~250 bp insert size)

  16. Preliminary round of sequencing M. occulta and M. oculata genes assemble together as splice variants

  17. Is the transcriptome assembly any good? • No reference genome! … • 96/98 Sanger-sequenced Molgulid genes found in assembly of individual lanes. • Can we distinguish & count parent transcripts in the hybrid? Query: 1 MDSSNRSHPNAHLQYHTDYNYPPFRRVMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH 60 MDSSNRSHPNAHLQYHTDYNYPPFR+VMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH Sbjct: 50 MDSSNRSHPNAHLQYHTDYNYPPFRKVMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH 229

  18. A large subset of transcripts have variation ~10% (at DNA level) Histogram Fraction bases identical in alignment

  19. We can count by allele!

  20. Hybrids express only M. occulta (tailless) versions??

  21. manx and p68/bobcat – probable isoform variation manx p68 RNA helicase manx Swalla et al. (1999) Development 126:1643-1653

  22. M. occulta(tailless) bobcat gene – expression by exon position in transcript

  23. Molgula – what’s next? • More time points for M. occulta; replicates. • Improve assembly (assemble all together). • Transcriptome-wide investigation of specific pathways (notochord, metamorphosis) • Transcriptome-wide search for pseudogenes in occulta (tailless), especially. • Sequencing genomes (PacBio?)

  24. Acknowledgements: The mRNAseq/k-mer gang: • Elijah Lowe • KanchanPavangadkar • LikitPreeyanon • Jason Pell • RosangelaCanino-Koning • ArendHintze Collaborators: • Billie Swalla & Max Maliska (UW/FHL) Funding: BEACON/NST STC; USDA NIFA; MSU, startup and iCER; DOE; Amazon Education.

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