Tail loss: investigating the Molgula

C. Titus BrownAsst Professor, CSE / MMGMichigan 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)

Station Biologique de Roscoff

Seven species of Molgula described by H. Lacaze-Duthiers and his students

(Swalla et al. found six of them)

http://www.sb-roscoff.fr/

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.

Molgula oculata notochord(40 cells, converged & extended)

Molgula occulta no notochord(20 cells, not converged & extended)

Hybrid notochord(20 cells, converged & extended)

Notochord Formation in Molgulids

Swalla and Jeffery, 1996

Takada et al. (2002) Evol. & Dev. 4:3 205-211

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

M. oculata M. occulta HybridsLarval muscle actin

gene expressionAbsent in tailless species

Tailed species gene is expressed in hybrids

Jeffery WR, Swalla BJ et al. (1999) Mol. Biol. Evol. 16(5):646-654.

In molgulid ascidians, manx and p68/bobcat are found in a gene complexand are required for tail development.

manx

manx

p68 RNA helicase

Swalla et al. (1999) Development 126:1643-1653

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?

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)

Approach

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

Allele specific expression

Deep, quantitative sampling of poly-A RNA population.

Can be used for expression analysis, transcriptome annotation (note, no reference!)

Short-read sequencing of mRNA

Overall protocol

Preliminary round of sequencing

(Illumina 76 bp x 2, ~250 bp insert size)

Sample Name Total reads after trim+filter

Loci (total genes, > 500)

Total incl splice

variants (> 500)

M. oculata (gastrula) 35,252,607 13,172 16,269

Hybrid (gastrula) 38,690,601 14,148 24,209M. occulta (gastrula) 22,548,831 8,046 10,802M. oculata (neurula) 38,030,938 10,365 11,043

Hybrid (neurula) 38,699,913 14,400 29,189M. oculata (tailbud) 38,073,640 14,204 17,835

Hybrid (tailbud) 34,307,907 15,399 26,594

Preliminary round of sequencing

Sample Name Total reads after trim+filter

Loci (total genes, > 500)

Total incl splice

variants (> 500)

M. oculata (gastrula) 35,252,607 13,172 16,269

Hybrid (gastrula) 38,690,601 14,148 24,209M. occulta (gastrula) 22,548,831 8,046 10,802M. oculata (neurula) 38,030,938 10,365 11,043

Hybrid (neurula) 38,699,913 14,400 29,189M. oculata (tailbud) 38,073,640 14,204 17,835

Hybrid (tailbud) 34,307,907 15,399 26,594

M. occulta and M. oculata genes assemble together as splice variants

No reference genome! …

96/98 Sanger-sequenced Molgulid genes found in assembly of individual lanes.

Can we distinguish & count parent transcripts in the hybrid?

Is the transcriptome assembly any good?

Query: 1MDSSNRSHPNAHLQYHTDYNYPPFRRVMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH 60 MDSSNRSHPNAHLQYHTDYNYPPFR+VMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEHSbjct: 50MDSSNRSHPNAHLQYHTDYNYPPFRKVMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH 229

A large subset of transcripts have variation ~10% (at DNA level)

Fraction bases identical in alignment

Hist

ogra

m

We can count by allele!

Sample NameM.

oculata

manx

M. occult

a manx

 M.

oculata

bobcat

M. occult

a bobcat

M. oculata (gastrula) 9 0 11 0Hybrid (gastrula) 0 28 0 255

M. occulta (gastrula) 0 41 0 76M. oculata (neurula) 5 0 4 1

Hybrid (neurula) 0 38 0 223M. oculata (tailbud) 6 0 6 0

Hybrid (tailbud) 0 8 0 121

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

Sample NameM.

oculata

manx

M. occult

a manx

 M.

oculata

bobcat

M. occult

a bobcat

M. oculata (gastrula) 9 0 11 0Hybrid (gastrula) 0 28 0 255

M. occulta (gastrula) 0 41 0 76M. oculata (neurula) 5 0 4 1

Hybrid (neurula) 0 38 0 223M. oculata (tailbud) 6 0 6 0

Hybrid (tailbud) 0 8 0 121

manx and p68/bobcat –probable isoform variation

manx

manx

p68 RNA helicase

Swalla et al. (1999) Development 126:1643-1653

0 1000 2000 3000 4000 5000 60000

200

400

600

800

1000

1200

occ/gastrulahyb/gastrula

M. occulta (tailless) bobcat gene – expression by exon

position in transcript

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?)

Molgula – what’s next?

Acknowledgements:The mRNAseq/k-mer

gang:

Elijah Lowe Kanchan Pavangadkar

Likit Preeyanon Jason Pell Rosangela Canino-Koning Arend Hintze

Collaborators:

Billie Swalla & Max Maliska (UW/FHL)

Funding: BEACON/NST STC; USDA NIFA; MSU, startup and iCER; DOE; Amazon Education.